Targeted Prevention of Human Ehrlichiosis

蜱传疾病的发病率在过去二十年中急剧上升，并继续上升。 由查菲埃里希体（Ehrlichia chaffeensis，Ech）引起的人单核细胞埃里希体病是最普遍的、终身的、 威胁性的、新出现的蜱传人畜共患病。Ech是一种专性胞内细菌， 立克次体治疗的选择是广谱抗生素强力霉素，这是有效的，只有当开始 早了目前还没有FDA批准的Ech疫苗。我们的长期目标是找到证据- 基于疫苗的方法，通过靶向立克次体的多个关键步骤有效保护人类 感染周期为了实现这一目标，我们鉴定了四种具有已知功能的Ech表面暴露蛋白 Ech存活所需的，并且也缺乏与人蛋白质的同源性，OMP-1/P28，进入触发蛋白 埃里希体（EtpE）和VirB 2。OMP-1/P28是免疫显性的表面暴露的外膜蛋白 其具有细菌获取营养所必需的孔蛋白活性。P28和OMP-1B主要是 分别在哺乳动物和蜱中表达。EtpE是一种侵袭素，其使用其C-末端（EtpE-C）结合 宿主细胞受体触发Ech进入。我们已经表明，IV型分泌系统（T4 SS）是必需的， 在宿主细胞内存活。VirB 2是T4 SS菌毛蛋白，是T4 SS机制的一部分。免疫 重组P28、EtpE或VirB 2蛋白的小鼠产生Ech-specific抗体应答， 防止感染。这些数据支持我们的前提，这些蛋白质作为合理的疫苗 用于靶向哺乳动物宿主细胞的Ech感染中的非重叠过程的候选物。DNA疫苗 提供了许多优于传统疫苗的潜在优势，包括刺激体液和 T细胞介导的应答，改进的疫苗稳定性，不存在任何感染因子，以及相对免疫原性。 易于封装多组件和大规模制造。我们展示了Ech DNA的可行性 通过经皮无针喷射注射DNA疫苗安全地免疫犬， 并证明了对DNA疫苗的体液和细胞介导的免疫应答。我们的假设是 用编码P28、OMP-1B、EtpE和VirB 2质粒DNA疫苗单独或联合免疫 防止Ech从蜱传播到哺乳动物。为了验证这一假设，我们的具体目标是：1。到 构建编码P28、OMP-1B、EtpE-C和VirB 2的DNA疫苗， 和细胞介导的免疫应答，并评估免疫小鼠对 用蜱细胞中培养的Ech感染。2.检测P28、OMP-1B、EtpE-C和VirB 2是否免疫犬 可以防止受感染的蜱传播Ech。拟议研究的直接结果将是 为Ech候选疫苗阻断Ech的DNA疫苗方法提供原理证明 从蜱虫到狗的传播长期的结果将是抗感染疫苗的发展 对人体内的HME不会造成不良影响。

The incidence of tick-borne diseases has risen dramatically in the past two decades, and continues to rise. Human monocytic ehrlichiosis caused by Ehrlichia chaffeensis (Ech) is one of the most prevalent, life- threatening, emerging tick-borne zoonoses in the US. Ech is an obligatory intracellular bacterium of the order Rickettsiales. Therapy of choice is the broad-spectrum antibiotic doxycycline, which is effective only if initiated early. Currently there is no FDA-approved vaccine for Ech. Our long-term goal is to develop an evidence- based vaccine approach to effectively protect humans by targeting multiple critical steps of the rickettsial infection cycle. Toward this goal, we identified four Ech surface-exposed proteins that have known functions required for Ech survival, and that also lack homology to human proteins, OMP-1/P28, Entry triggering protein of Ehrlichia (EtpE), and VirB2. OMP-1/P28s are immunodominant surface-exposed outer membrane proteins that have porin activity essential for bacterial nutrient acquisition. P28 and OMP-1B are predominantly expressed in mammals and ticks, respectively. EtpE is an invasin that uses its C-terminus (EtpE-C) to bind the host cell receptor to trigger Ech entry. We have shown that the type IV secretion system (T4SS) is essential for Ech survival within the host cell. VirB2 is a T4SS pilus protein that is part of the T4SS machinery. Immunization of mice with recombinant P28, EtpE, or VirB2 proteins generated Ech-specific antibody responses that prevented Ech infection. These data support our premise that these proteins serve as rational vaccine candidates for targeting non-overlapping processes in Ech infection of mammalian host cells. DNA vaccines offer a number of potential advantages over traditional vaccines, including the stimulation of both humoral and T-cell-mediated responses, improved vaccine stability, the absence of any infectious agent, and the relative ease of packaging multi-components and large-scale manufacture. We showed the feasibility of an Ech DNA vaccine in dogs by safely immunizing dogs with the DNA vaccines by percutaneous needle-free jet injection and demonstrating humoral and cell-mediated immune responses to the DNA vaccines. Our hypothesis is immunization with plasmid DNA vaccine encoding P28, OMP-1B, EtpE and VirB2 singly or in combination prevents Ech transmission from ticks to mammals. To test this hypothesis, our Specific Aims are: 1. To construct DNA vaccines encoding P28, OMP-1B, EtpE-C, and VirB2, determine the development of humoral and cell-mediated immune responses in immunized mice, and evaluate protection of immunized mice from infection with Ech cultured in tick cells. 2. To test if immunization of dogs with P28, OMP-1B, EtpE-C and VirB2 can prevent Ech transmission from infected ticks. The immediate outcomes of the proposed studies will be to provide proof-of-principle for a DNA vaccine approach to the Ech vaccine candidates for blocking of Ech transmission from ticks to dogs. The long-term outcome will be development of an anti-infective vaccine against HME in humans that does not cause adverse effects.