High throughput high resolution profiling of antibody specificities of polyclonal

描述(由申请人提供)：艾滋病毒会在感染者体内产生抗体。虽然对大多数人来说，这种反应对感染的特定病毒分离株具有高度特异性，但也有一些人安装了广泛交叉的中和抗体，似乎可以控制病毒。据推测，这种交叉中和活性是抗击艾滋病的核心，并将是预防性艾滋病疫苗的必要属性。因此，能够批判性地分析针对HIV的抗体反应并描述与病毒中和相关的特异性是非常必要的。然而，目前的血清学方法繁琐、费力且分辨率不令人满意，对HIV包膜大块的抗体特异性进行分类。在这里，我们提出了一种新的、经过大幅改进的技术平台，它可以相对容易、低成本和高通量地生成非常高分辨率的多克隆血清抗体特异性图谱。这项技术的本质是两种新方法的结合：一种是由长度从50到300个氨基酸不等的HIV包膜多肽/结构域组成的全面阵列的噬菌体展示文库，另一种是独特的载体系统，它可以将噬菌体展示转化为与第二代DNA循环测序兼容的展示。总而言之，这种方法被称为结构域扫描文库的深度平移，使人们能够分析选定的多克隆血清的完整抗体库。生成的数据输出由与HIV包膜片段相对应的数十万个亲和分离肽组成。在这项研究中，将使用一组&gt；400个具有良好特征的HIV感染者的血清样本来测试这些方法的可行性以及它们的实用性和生产力的评估，这些样本的中和能力从不存在/弱到非常有效(占集合的前2%)。选定的血清将受到 对深度平移和待生成的数据集进行分类，实现机器学习的计算方法。这些将产生与艾滋病毒中和最相关的表位的概况。这些信息对于我们理解HIV的中和机制以及指导合理的疫苗设计都是非常有用的。此外，这项初步研究的可行性证明将作为更详细和全面的研究的基础，目的是更好地了解艾滋病毒与免疫、疾病进展和预防艾滋病的相关性。 公共卫生相关性：将通过对区域扫描库的深度平移来分析HIV感染者的多克隆血清的综合小组，这是一种产生高分辨率抗体特异性简档的新方法。具体的重点将是阐明艾滋病毒中和的相关因素以及这一活动与病毒包膜的特定表位的关联。这些病毒中和相关性的发现将有助于合理设计艾滋病疫苗，更好地了解疾病的发展和预防。

DESCRIPTION (provided by applicant): HIV elicits the production of antibodies in the infected individual. Whereas for most, this response is highly specific for the particular viral isolate of infection, there are some individuals that mount broadly cross neutralizing antibodies that appear to keep the virus in check. It is assumed that such cross neutralizing activity is central i combating AIDS and would be a necessary attribute of a prophylactic AIDS vaccine. Therefore, it is highly desirable to be able to critically analyze the antibody response towards HIV and profile the specificities correlated with virus neutralization. Current serological methods, however, are tedious, laborious and of unsatisfactory resolution, classifying antibody specificities to gross chunks of the HIV envelope. Here we propose a novel and much improved technological platform that generates exceptionally high resolution profiles of the antibody specificities of polyclonal serum with relative ease, low cost and at high throughput. The essence of the technology is the combination of two new methods; a phage display library of a comprehensive array of HIV envelope peptides/domains ranging in length from 50 to 300 amino acids, and a unique vector system that converts phage display compatible with second generation DNA cyclic sequencing. Together, this methodology, coined Deep Panning of Domain Scan libraries, enables one to analyze the complete antibody repertoire of selected polyclonal sera. The data output generated consists of hundreds of thousands of affinity isolated peptides corresponding to HIV envelope segments. In this study feasibility of the methods and evaluation of their utility and productivity will be tested using a panel of >400 well characterize serum samples of HIV infected individuals that range in their neutralizing capacity from non-existent/weak to extraordinarily potent (top 2% of the collection). Selected sera will be subjected to Deep Panning and the data sets to be generated will be classified implementing machine learning computational methods. These will produce a profile of those epitopes that best correlate with HIV neutralization. It is proposed that this information will be extremely useful fo our understanding of mechanisms of HIV neutralization and as guides to rational vaccine design. Moreover, the proof of feasibility in this pilot study will serve as the basis for more elaborate and comprehensive studies targeted to better understand HIV correlates of immunity, disease progression and prevention of AIDS. PUBLIC HEALTH RELEVANCE: A comprehensive panel of polyclonal sera of HIV infected individuals will be analyzed by Deep Panning of Domain Scan libraries, a novel methodology that generates high resolution profiles of antibody specificities. Specific focus will be on elucidation of the correlates of HIV neutralization and association of this activity to specific epitopes of the viral envelope. The discovery of these correlates of virus neutralization will be useful for rational design of AIDS vaccines and better understanding of disease progression and prevention.