Targeted Prevention of Human Ehrlichiosis

在过去的二十年里，壁虱传播疾病的发病率急剧上升，而且还在继续上升。 由查菲埃立克体病引起的人类单核细胞埃立克体病是最常见的一种生命- 在美国，新出现的具有威胁性的壁虱传播的人畜共患病。ECH是一种必须的胞内细菌。 Rickettsiales。选择的治疗方法是广谱抗生素多西环素，只有在开始治疗时才有效 很早。目前还没有FDA批准的Ech疫苗。我们的长期目标是找到证据- 针对立克次体多个关键步骤有效保护人类的疫苗方法 感染周期。为了实现这一目标，我们确定了四种具有已知功能的Ech表面暴露蛋白。 Ech生存所必需的，而且与人类蛋白OMP-1/P28也缺乏同源性，进入触发蛋白 EhrlichiaEtpE；OMP-1/P28s是免疫优势的表面暴露外膜蛋白 具有对细菌营养获取至关重要的孔蛋白活性。P28和OMP-1B主要是 分别在哺乳动物和扁虱体内表达。EtpE是一种侵入素，它使用其C末端(EtpE-C)与 宿主细胞受体触发Ech进入。我们已经证明，IV型分泌系统(T4SS)对于 ECH在宿主细胞内存活。VirB2是一种T4SS菌毛蛋白，是T4SS机制的一部分。免疫接种 重组P28、EtpE或VirB2蛋白的小鼠产生了Ech特异的抗体反应 预防了Ech感染。这些数据支持我们的假设，即这些蛋白质是合理的疫苗。 在哺乳动物宿主细胞的Ech感染中靶向非重叠过程的候选。DNA疫苗 与传统疫苗相比，提供了许多潜在的优势，包括刺激体液和 T细胞介导的反应，提高了疫苗的稳定性，没有任何感染源，以及相关的 易包装、多部件、大批量制造。我们展示了Ech DNA的可行性 经皮无针注射DNA疫苗安全免疫犬的研究 并展示了对DNA疫苗的体液和细胞免疫反应。我们的假设是 编码P28、OMP-1B、EtpE和VirB2的DNA疫苗单独或联合免疫 防止Ech从扁虱传播到哺乳动物。为了验证这一假设，我们的具体目标是：1. 构建编码P28、OMP-1B、EtpE-C和VirB2的DNA疫苗，确定体液的发育 和细胞介导的免疫反应，并评价免疫小鼠对 在硬蜱细胞中培养的Ech感染。2.检测P28、OMP-1B、EtpE-C和VirB2对犬的免疫效果 可以防止受感染的扁虱传播Ech。拟议研究的直接结果将是： 为阻断Ech的候选Ech疫苗提供DNA疫苗方法的原理证据 从扁虱传染给狗。长期的结果将是开发一种抗感染疫苗 在人体内对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.