Inhibition of Ehrlichial Infection by Intracellular Nanobody

细胞内纳米抗体抑制埃利希体感染

• 批准号: 9808090
• 负责人: YASUKO RIKIHISA
• 金额: $ 23.4万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-04 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808090
• 关键词: Amino Acid Sequence; Anaplasmosis; Antibodies; Antigen Targeting; Antigens; Bacteria; Basic Science; Binding; Biological; Cells; Centers for Disease Control and Prevention (U.S.); Cloning; Communicable Diseases; Complementary DNA; Cytoplasm; Cytosol; Data; Development; Disease; Drug Kinetics; Ehrlichia chaffeensis; Ehrlichiosis; Emerging Communicable Diseases; Environment; Essential Genes; Fab Immunoglobulins; Fever; Future; Goals; Green Fluorescent Proteins; Heterogeneous-Nuclear Ribonucleoprotein K; Human; Immunize; In Vitro; Infection; Inflammatory; Knock-out; Knowledge; Libraries; Life; Link; Llama; Lymphocyte; Malignant Neoplasms; Measures; Messenger RNA; Methods; Mission; Modeling; Molecular; Mus; Organism; Outcome; Peptide Nucleic Acids; Peptide antibodies; Peptides; Phage Display; Pharmaceutical Preparations; Pilot Projects; Plasmids; Precision therapeutics; Prevalence; Production; Proteins; Public Health; Receptor Cell; Research; Rickettsia; Signal Pathway; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic Human Experimentation; Therapeutic Intervention; Toxic effect; Toxin; Transfection; Translational Research; Type IV Secretion System Pathway; Typhus; United States National Institutes of Health; Vector-transmitted infectious disease; Virulence Factors; Work; antigen binding; disability; effective therapy; efficacy testing; expression vector; in vivo; innovation; knock-down; monocyte; mutant; nanobodies; novel; novel strategies; pathogen; prevent; prophylactic; tool

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.

立克次氏体引起危及生命的病媒传播疾病，包括无形体病和埃里希体病， 在世界范围内的流行率大大增加。发展中国家面临两大障碍， 对由这些专性细胞内细菌引起的疾病的有效治疗和预防措施：1） 重要的疾病相关的细菌和宿主分子是细胞内的，受到直接抗体的保护 (Ab)或药物攻击，以及2）细菌特异性致命目标难以识别，因为常规 诸如敲除专性细胞内感染必需基因的突变体的方法是不可行的。 查菲埃里希体（Ehrlichia chaffeensis，Ech）就是一个典型的例子。IV型分泌系统（T4 SS）在所有 是立克次体的病原体，也是宿主感染所必需的。Ech T4 SS效应子的最新阐明 埃里希体易位因子1和2（Etf-1和Etf-2）为创新的分子生物学提供了关键靶点。 接近。纳米抗体是源自重链的最小的完整抗原结合片段（VHH）。 只有骆驼科动物体内有抗体纳米抗体是蛋白水解稳定的，并且在还原的细胞中具有生物活性。 因此，它们作为Ech感染的治疗剂具有更大的潜力， 研究工具比传统的抗体。VHH可以克隆到哺乳动物表达质粒中，并且 在异源系统中以高水平在细胞内表达。事实上，我们显示VHH的转染， 结合人异质核核糖核蛋白K可以阻断Ech感染人细胞。的目标 拟议的研究是进行一项试点研究，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抗体。我们将通过两个目标来实现这一目标。目标1是 为了分离和表征抑制Ech感染的Etf-1和Etf-2特异性iAb，通过：1）从大肠杆菌中克隆VHH cDNA， 将Etf-1和Etf-2免疫的美洲驼淋巴细胞mRNA导入噬菌体展示文库， 2）将Etf-1和Etf-2特异性VHH克隆到哺乳动物表达载体中， 3）鉴定Ech感染阻断性iAb并测定其氨基 酸序列;和4）分析iAb抑制Etf-1、Etf-2功能和阻断Ech的机制 感染目的2是将选择的与细胞穿透肽缀合的VHH肽（VHH-CPP）递送至 通过以下方式阻断Ech感染：1）优化Ech感染阻断VHH-CPP进入人细胞的细胞内递送 2）测试所选VHH-CPP在体外抑制Ech感染的功效;和3）分析 VHH-CPP在小鼠中抑制Ech感染的药代动力学、毒性和功效。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， 以及对强制性细胞内感染的转化研究。最终，我们的发现将提供 这是一种新的抗立克次体治疗方法的重要科学依据。