Identification of the mechanism of protection against a B. burgdorferi protein CspZ for the prevention of Lyme disease

项目总结： 莱姆病(LD)是北半球最常见的媒介传播疾病。这种疾病是由 由伯氏疏螺旋体(BBSL)引起，它从扁虱叮咬到皮肤传播到不同的 组织，导致关节炎、心脏炎和神经疏螺旋体病。目前还没有有效的预防措施。我们的目标 是了解保护性免疫的机制，以开发安全有效的预防工具 人类身份。补体是血液中一种重要的先天防御机制，可由多个 途径包括由抗体-抗原复合体诱导的经典途径和替代途径 途径，这是由补体C3b蛋白与微生物表面结合而触发的。这个 补体的激活会在细菌表面形成一种成孔复合体C5b-9，从而导致溶解。在……里面 在没有病原体的情况下，补体被补体调节剂抑制，以避免宿主细胞的损害。为 例如，因子H(FH)专门抑制替代途径。螺旋体产生一个外表面 CspZ蛋白广泛存在于BBSL物种中，可有效地传递给哺乳动物宿主。CSPZ是 当细菌进入这些宿主时产生的，并通过将FH聚集到其表面来促进BBSL的传播 抑制补体介导的杀伤。然而，CspZ的免疫既不能诱导高水平的 杀菌抗体也不能保护小鼠免受BBSL的侵袭。一种可能性是CSPZ的保护性 表位被FH饱和，这将不允许该蛋白诱导足够的抗体来有效地 体内消除BBSL。因此，我们产生了一个FH结合缺陷的CspZ-Y207A/Y211A突变体(CspZ-YA)， 导致这种蛋白质的FH结合位点上的表位暴露。我们发现CspZ-YA而不是CspZ 接种疫苗可保护小鼠免受BBSL感染的侵袭。我们证明了被动免疫 来自CspZ-YA-而不是CspZ免疫的小鼠的抗体中的一种可保护小鼠免受BBSL定植。我们 观察到，前一种抗体可阻断FH与CspZ的结合，但不能阻断后者。这些发现表明CspZ- YA的FH结合部位暴露，可诱导保护性抗体促进BBSL清除。因此， 总体目标是确定CspZ-YA疫苗的保护机制。我们假设 CspZ-YA疫苗的保护性抗体阻止FH依赖的替代途径的逃避和 促进经典通路的激活，导致BBSL的杀伤。为了检验这一假设，我们将1)评估 CspZ-YA疫苗诱导的抗体在促进经典性和替代性免疫中的作用 补体途径，2)确定CspZ-YA诱导的抗体导致BBSL清除的机制。 这些研究将阐明CspZ-YA作为有效疫苗发挥作用的机制。是这样的 机制将为进一步确定该抗原的保护性表位以识别有效的 作为LD预防制剂的单抗。总体而言，这些信息最终将提供有效的 预防BBSL感染和减轻人类LD负担的策略。

Project Summary: Lyme disease (LD) is the most common vector-borne disease in the northern hemisphere. The disease is caused by the spirochete Borrelia burgdorferi sensu lato (Bbsl), which spreads from a tick bite to the skin to different tissues, leading to arthritis, carditis, and neuroborreliosis. No effective prevention is currently available. Our goal is to understand the mechanisms of protective immunity to develop safe and potent prevention tools against human LD. Complement is an important innate defense mechanism in the blood that can be triggered by multiple pathways including the classical pathway, which is induced by antibody-antigen complexes, and the alternative pathway, which is triggered by the binding of the complement C3b protein with the microbial surface. The activation of complement results in a pore-forming complex, C5b-9, on the bacterial surface leading to lysis. In the absence of pathogens, complement is inhibited by complement regulators to avoid host cell damages. For example, Factor H (FH) specifically inhibits the alternative pathway. Spirochetes produce an outer surface protein, CspZ, widely present in Bbsl species that can be efficiently transmitted to mammalian hosts. CspZ is produced when bacteria enter these hosts and facilitates Bbsl dissemination by recruiting FH to its surface thus inhibiting complement-mediated killing. However, immunization of CspZ neither induces great levels of bactericidal antibodies nor does it protect mice from Bbsl colonization. One possibility is that CspZ’s protective epitopes are saturated by FH, which would not allow this protein to induce sufficient antibodies to efficiently eliminate Bbsl in vivo. We thus generated a CspZ-Y207A/Y211A mutant (CspZ-YA) that is deficient in FH-binding, leading to the exposure of the epitopes on this protein’s FH-binding sites. We found that CspZ-YA but not CspZ vaccination protects mice from Bbsl colonization via tick infection. We demonstrated that passive immunization of the antibodies from CspZ-YA- but not CspZ-immunized mice protects mice from Bbsl colonization. We observed that the former but not later antibodies block FH binding to CspZ. These findings suggest that CspZ- YA’s FH-binding site is exposed, which could induce protective antibodies to promote Bbsl clearance. Thus, the overall objective is to define the protective mechanisms of the CspZ-YA vaccine. We hypothesize that the protective antibodies from CspZ-YA vaccination block FH-dependent evasion of the alternative pathway and promote activation of the classical pathway, resulting in Bbsl killing. To test the hypothesis, we will 1) assess the role of the antibodies induced by CspZ-YA vaccination in promoting the activation of classical and alternative complement pathways, 2) define the mechanisms of the CspZ-YA-induced antibodies leading to Bbsl clearance. These studies will elucidate the mechanisms that allow CspZ-YA to function as an effective vaccine. Such mechanisms will build the foundation to further identify the protective epitopes of this antigen to identify potent monoclonal antibodies as LD prophylactic agents. Overall, this information will ultimately provide effective strategies to prevent Bbsl infection and reduce the burden of human LD.