Predicting Protein Evolution with Phage Escape

描述(由申请人提供)：鉴于人类感染禽流感病毒株的极端死亡率，出现大流行性流感疫情令人极为关注。大流行毒株被认为是通过在与多种流感病毒混合感染期间对病毒基因组片段进行重新分类，以及逐渐积累足以改变特定毒株的抗原性的突变，从而使先前存在的宿主免疫变得无效(抗原漂移)，从而引入遗传上不同的蛋白质序列而发生的。该项目的目标是通过使用无偏见的组合文库方法在体外反映这些过程，从而允许访问大段抗原空间。具体地说，我们将利用我们实验室开发的噬菌体逃逸技术来生成病毒表面糖蛋白血凝素(HA)的组合库，以便访问流感病毒可用的序列空间。我们策略的实质是一种交替筛选人类抗体的方案，首先阻止HA与细胞结合，然后选择突变的HA蛋白，这些突变的HA蛋白“逃脱”我们的抗体并保持与细胞的结合。重复这一过程将揭示一系列不同的HA克隆能够与细胞结合，以及与每一种HA相对应的一组中和抗体。最终，我们设想我们的方法将允许对目前的禽流感病毒株(H5)、过去发生的毒力强的HA突变株(例如H1)以及尚未出现的禽流感病毒株进行评估。此外，我们的实验设计所固有的是同时鉴定特定的人类抗体，这些抗体中和了HA与宿主细胞的结合。与公共卫生相关：很少有一年的流感不是公共卫生问题，尤其是近年来禽流感大流行的威胁在公众心中引起了极大的焦虑。我们的建议利用现代分子生物学技术来模拟感染所需的流感蛋白的进化。我们的目标是建立一个能够预测流感病毒株的实验室系统，并在这个过程中，在这些病毒株可能对公共健康构成威胁之前，为它们开发中和疗法。

DESCRIPTION (provided by applicant): In light of the extreme mortality rates associated with human infection with avian influenza strains, the emergence of a pandemic influenza outbreak is of immense concern. Pandemic strains are thought to occur through the introduction of genetically divergent protein sequences by reassortment of viral genomic segments during coinfection with multiple influenza viruses (antigenic shift), as well as gradual accumulation of mutations sufficient to alter the antigenicity of a given strain such that preexisting host immunity becomes ineffective (antigenic drift). The goal of this project is to mirror these processes in vitro by using an unbiased combinatorial library approach, thereby allowing access to large sections of antigenic space. Specifically, we will utilize phage escape technologies developed in our laboratory to generate combinatorial libraries of the viral surface glycoprotein hemagglutinin (HA) to allow access to the sequence space available to the influenza virus. The essence of our tactic is an alternating scheme of screening for human antibodies which first prevent HA binding to cells, followed by a selection for mutant HA proteins that "escape" our antibodies and remain bound to cells. Repeated iteration of this process will reveal an array of varying HA clones capable of binding to cells, as well as the set of neutralizing antibodies corresponding to each HA. Ultimately, we envision that our approach will allow for an assessment of both the current avian influenza strain (H5), virulent HA mutants which occurred in the past (e.g., H1), as well as those that have not yet emerged. Furthermore, inherent to our experimental design is a concurrent identification of specific human antibodies that neutralize HA binding to host cells. PUBLIC HEALTH RELEVANCE: Rarely does a year go by that influenza is not a public health concern, and in particular, the threat of a pandemic avian influenza in recent years has caused immense anxiety in the minds of the public. Our proposal utilizes modern molecular biology techniques to mimic the evolution of the influenza protein required for infection. We aim to generate a laboratory system that can predict influenza strains and in the process, develop neutralizing therapies for these strains before they can become a threat to public health.