Inhibition of Ehrlichial Infection by Intracellular Nanobody

立克次体引起威胁生命的病媒传播疾病，包括无浆体病和埃立克次体病，这些疾病是 在全球范围内的流行率大大增加。要取得进展，有两大障碍 这些必需性胞内细菌引起的疾病的有效治疗和预防措施：1) 重要的与疾病相关的细菌和宿主分子是细胞内的，不受直接抗体的影响 (AB)或药物攻击，以及2)细菌特有的致命目标很难识别，因为传统的 对强制性细胞内感染的必要基因进行基因敲除突变等方法是不可行的。 查菲埃利希氏菌(Ehrlichiachaffesis(Ech))就是一个典型的例子。IV型分泌系统(T4SS)在ALL中保守 立克次体，对宿主感染是必不可少的。Ech T4SS效应器的研究进展 埃立克易位因子1和2(ETF-1和ETF-2)为创新分子提供关键靶点 接近了。纳米体是最小的完整的抗原结合片段(VHH)，源于重链- 只有骆驼体内的抗体。纳米小体在蛋白质降解方面是稳定的，在还原后具有生物活性 细胞内环境；因此，它们作为Ech感染和治疗药物的潜力更大 研究工具比常规抗体更好。VHH可以克隆到哺乳动物表达载体中，并且 在异源系统中高水平在细胞内表达。事实上，我们证明了VHH的转染法 结合人异源核糖核蛋白K可以阻断Ech在人细胞中的感染。的目标是 建议的研究是开展一项初步研究，以开发和表征Ech感染阻断 用于未来治疗应用的细胞内纳米体。我们将通过两个目标实现这一目标。目标1是 通过以下方法分离和鉴定ETF-1和ETF-2特异性抑制Ech感染的IAbs：1)克隆VHH基因 ETF-1和ETF-2免疫骆驼淋巴细胞的文库构建及ETF-1和ETF-2的分离 2)将ETF-1和ETF-2特异的VHH克隆到哺乳动物表达系统中 载体转化产生IAbs；3)鉴定Ech感染阻断IAbs并测定其氨基酸 4)分析IAB抑制ETF-1、-2功能和阻断Ech的机制(S) 感染。目的2是将选定的与细胞穿透肽结合的VHH多肽(VHH-CPP)输送到 通过以下方式阻断Ech感染：1)优化Ech感染的细胞内传递--阻断VHH-CPP进入人细胞 2)体外检测VHH-CPP对Ech感染的抑制作用；3)分析 VHH-CPP在小鼠体内的药代动力学、毒性和抑制Ech感染的有效性。我们的结果将 展示了纳米体技术和CPP的新用途，以克服当前的障碍，推进基础设施 以及关于强制性细胞内感染的翻译研究。最终，我们的发现将提供 新的抗立克次体治疗方法的重要科学依据。

Rickettsia cause life-threatening vector-borne diseases, including Anaplasmosis and Ehrlichiosis, which are greatly increasing in worldwide prevalence. There are two major barriers to progress for the development of effective therapies and prophylactic measures for diseases caused by these obligatory intracellular bacteria: 1) the important disease-associated bacterial and host molecules are intracellular, protected from direct antibody (Ab) or drug attack, and 2) bacteria-specific lethal targets are difficult to identify because conventional approaches, such as knockout mutants of essential genes for obligatory intracellular infection, are not feasible. Ehrlichia chaffeensis (Ech) is a classic example. The Type IV Secretion System (T4SS) is conserved among all Rickettsial organisms and is essential for host infection. The recent elucidation of the Ech T4SS effectors Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) provide critical targets for innovative molecular approaches. Nanobodies are the smallest intact antigen-binding fragments (VHH) derived from heavy chain- only antibodies in camelids. Nanobodies are proteolytically stable and biologically active in reduced intracellular environments; therefore, they have greater potential as therapeutic agents in Ech infection and research tools than conventional antibodies. VHH can be cloned into a mammalian expression plasmid, and expressed intracellularly at high levels in heterologous systems. Indeed, we showed transfection of VHH that binds human heterogeneous nuclear ribonucleoprotein K can block Ech infection in human cells. The goal of the proposed research is to carry out a pilot study to develop and characterize Ech infection-blocking intracellular nanobodies for future therapeutic application. We will achieve this goal through two aims. Aim 1 is to isolate and characterize Etf-1 and Etf-2-specific iAbs that inhibit Ech infection by: 1) cloning VHH cDNA from Etf-1 and Etf-2-immunized llama lymphocyte mRNA into a phage display library and isolating Etf-1 and Etf-2 antigen-specific VHHs by panning; 2) cloning Etf-1 and Etf-2-specific VHHs into a mammalian expression vector to produce iAbs by transfection; 3) identifying Ech infection-blocking iAbs and determining their amino acid sequences; and 4) analyzing the mechanism(s) by which the iAbs inhibit Etf-1, -2 functions and block Ech infection. Aim 2 is to deliver selected VHH peptides conjugated with cell penetrating peptide (VHH-CPP) to block Ech infection by: 1) optimizing intracellular delivery of Ech infection-blocking VHH-CPP into human cells in vitro; 2) testing the efficacy of selected VHH-CPP for inhibition of Ech infection in vitro; and 3) analyzing VHH-CPP pharmacokinetics, toxicity, and efficacy for inhibiting Ech infection in mice. Our results will demonstrate the novel use of nanobody technology and CPP to overcome current barriers to advance basic and translational research on obligatory intracellular infection. Eventually, our findings will provide the important scientific basis for a novel anti-rickettsial therapeutic approach.