HOST RANGE DIVERSITY OF BACTERIOPHAGE FOR Y. PESTIS

描述(申请人提供)：鼠疫耶尔森氏菌，负责鼠疫的细菌，是一种A类病原体，是一种重要的生物防腐剂。如果被武器化，它可以很容易地直接进入呼吸系统，导致肺炎鼠疫迅速死亡。肺鼠疫的致死率超过50%，传染性很高，如果细菌被设计成抵抗抗生素，基本上是不可战胜的。作为对这种威胁的对策，我们建议开发一个针对鼠疫杆菌的噬菌体文库，可以高效地杀死这种细菌的多种菌株。AvidBiotics拥有独家获得技术来产生具有高度多样化尾巴的噬菌体家族，能够与突变形式的细菌结合，如波氏杆菌。在这一应用中，我们建议开发一个噬菌体文库来结合、感染和杀死突变的鼠疫杆菌，预测潜在的突变进入细菌中，以改变它们对抗生素或噬菌体表面受体的敏感性。这项技术是基于一系列产生多样性的逆转录元件(DGR)的发现，这些逆转录元件的功能是改变DNA序列及其编码的蛋白质。这些DGR可以被工程化为噬菌体，使噬菌体尾部纤维准确的受体结合部位的氨基酸序列多样化，从而产生一个具有超过1万亿条不同尾巴的噬菌体文库，这些尾巴可能针对鼠疫杆菌细胞表面受体。通过将这种噬菌体文库暴露于突变的或武器化的鼠疫细菌，能够感染、繁殖和杀死细菌的噬菌体可以被识别、选择、分离、扩增、雾化，然后部署以预防或治疗武器化鼠疫菌的感染。本申请的目的是展示利用DGR-系统精确和广泛地使噬菌体尾部纤维序列多样化的能力，作为一种创造具有广泛鼠疫嗜性的新型噬菌体的手段。这些研究将为未来的研究提供基础，以产生一个适当的、高度多样化的噬菌体池，作为有效对抗武器化鼠疫细菌的可靠、多产的诊断和治疗剂来源。我们预计，第二阶段的后续项目将需要开发一种工艺，以生产和配制针对鼠疫杆菌的多样化噬菌体文库的代表性成员，并进行临床前安全性研究，以准备一个快速反应的反恐系统来管理人类的肺炎鼠疫。

DESCRIPTION (provided by applicant): Yersinia pestis, the bacterium responsible for plague, is a Category A pathogen, a significant biowarfare agent. If weaponized, it can easily gain direct access to the respiratory system to cause rapid death by pneumonic plague. Pneumonic plague has fatality rates over 50%, is highly contagious, and if the bacteria are engineered to resist antibiotics, is essentially unbeatable. As a countermeasure to such a threat, we propose to develop a library of bacteriophages ("phages") specific for Y. pestis that can kill multiple strains of this bacterium in a highly efficient manner. AvidBiotics has exclusive access to technology to generate families of phage with highly diverse tails capable of binding to mutant forms of bacteria, such as Bordetella. In this application, we propose to develop a library of phage to bind to, infect and kill mutant Y. pestis bacteria, anticipating potential mutations engineered into the bacteria to change their sensitivities to antibiotics or surface receptors for phage. This technology is based on the discovery of a family of diversity-generating retroelements (DGRs) that function to vary DNA sequences and the proteins they encode. These DGRs can be engineered into phages to diversify specifically the amino acid sequence of the precise receptor-binding site of the phage tail fiber, thereby generating a library of phages with more than a trillion different possible tails targeting Y. pestis cell surface receptors. By exposing such a phage library to mutant or weaponized plague bacteria, phages capable of infecting, multiplying and killing the bacteria could be identified, selected, isolated, amplified, aerosolized and then deployed to protect against or treat infections by weaponized Y. pestis. The objective of this application is to demonstrate the ability to utilize the DGR-system to precisely and extensively diversify the phage tail fiber sequences as a means to create novel phage with broad Y. pestis tropism. These studies will provide the foundation for future studies to generate a suitable, formulated pool of highly diverse phages that will serve as a reliable, productive source of diagnostic and therapeutic agents effective against weaponized plague bacteria. We anticipate that a Phase II follow-on project would entail developing a process to GLP manufacture and formulate representative members of the diverse phage library targeting Y. pestis and conduct pre-clinical safety studies to prepare a rapid response counter- terrorism system to manage pneumonic plague in humans.