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

项目概要： 莱姆病是北方最常见的病媒传播疾病。这种疾病是由 由螺旋体伯氏疏螺旋体广义（Bbsl），从蜱叮咬皮肤传播到不同的 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 了解保护性免疫的机制，以开发安全有效的预防工具， 人LD。补体是血液中一种重要的先天防御机制，可由多种免疫反应触发。 途径包括经典途径，其由抗体-抗原复合物诱导，以及替代途径。 通过补体C3 b蛋白与微生物表面的结合而触发。的 补体的活化导致细菌表面上的孔形成复合物C5 b-9，从而导致裂解。在 在没有病原体的情况下，补体被补体调节剂抑制，以避免宿主细胞损伤。为 例如，因子H（FH）特异性抑制旁路途径。螺旋体产生一个外表面 CspZ蛋白广泛存在于Bbsl物种中，可有效地传播至哺乳动物宿主。CspZ是 当细菌进入这些宿主时产生，并通过将FH募集到其表面来促进Bbsl传播， 抑制补体介导的杀伤。然而，CspZ的免疫既不诱导高水平的免疫应答， 也不能保护小鼠免受Bbsl定殖。一种可能是CspZ的保护性 表位被FH饱和，这将不允许该蛋白质诱导足够的抗体以有效地 在体内消除Bbsl。因此，我们产生了FH结合缺陷的CspZ-Y207 A/Y211 A突变体（CspZ-YA）， 导致该蛋白质的FH结合位点上的表位暴露。我们发现CspZ-YA而不是CspZ 疫苗接种保护小鼠免于经由蜱感染的Bbsl定殖。我们证明了被动免疫 来自CspZ-YA免疫的小鼠而不是CspZ免疫的小鼠的抗体保护小鼠免于Bbsl定殖。我们 观察到前者而不是后者的抗体阻断FH与CspZ的结合。这些发现表明CspZ- YA的FH结合位点暴露，这可以诱导保护性抗体以促进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 来自CspZ-YA疫苗接种的保护性抗体阻断旁路途径的FH依赖性逃避， 促进经典途径的活化，导致Bbsl杀伤。为了验证假设，我们将1）评估 CspZ-YA疫苗接种诱导的抗体在促进经典和替代激活中的作用 补体途径，2）定义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。等 机制的研究将为进一步鉴定该抗原的保护性表位奠定基础， 单克隆抗体作为LD预防剂。总的来说，这些信息最终将提供有效的 预防Bbsl感染和减少人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.