High throughput high resolution profiling of antibody specificities of polyclonal

描述（由申请方提供）：HIV促使受感染个体产生抗体。虽然对于大多数人来说，这种反应对特定的病毒分离株感染具有高度特异性，但也有一些人安装了广泛的交叉中和抗体，似乎可以控制病毒。假定这种交叉中和活性是对抗AIDS的核心，并且是预防性AIDS疫苗的必要属性。因此，非常希望能够批判性地分析针对HIV的抗体应答，并分析与病毒中和相关的特异性。然而，目前的血清学方法是繁琐的，费力的和不令人满意的分辨率，分类抗体特异性的总块的HIV包膜。在这里，我们提出了一种新的和大大改进的技术平台，产生异常高分辨率的多克隆血清的抗体特异性的配置文件，相对容易，低成本和高通量。该技术的本质是两种新方法的结合;一种是长度为50至300个氨基酸的HIV包膜肽/结构域的全面阵列的噬菌体展示文库，另一种是将噬菌体展示转化为与第二代DNA循环测序兼容的独特载体系统。总之，这种方法，创造了域扫描文库的深度淘选，使人们能够分析选定的多克隆血清的完整抗体库。生成的数据输出由数十万个与HIV包膜片段相对应的亲和分离肽组成。在本研究中，将使用一组>400个良好表征的HIV感染个体的血清样品来测试方法的可行性及其效用和生产力的评估，所述HIV感染个体的中和能力从不存在/弱到非常强（收集的前2%）。将对选定的血清进行 到深度平移，并且要生成的数据集将被分类，实现机器学习计算方法。这些将产生与HIV中和最相关的那些表位的概况。建议，这些信息将是非常有用的，我们了解艾滋病毒中和的机制，并作为指导，合理的疫苗设计。此外，这项试点研究的可行性证明将作为更详细和全面研究的基础，以更好地了解艾滋病毒与免疫力、疾病进展和艾滋病预防的相关性。 公共卫生相关性：将通过域扫描文库的深度淘选分析HIV感染个体的多克隆血清的综合组，这是一种生成抗体特异性的高分辨率谱的新方法。具体的重点将是阐明HIV中和的相关性和这种活性与病毒包膜的特定表位的关联。这些病毒中和相关因素的发现将有助于合理设计艾滋病疫苗，更好地了解疾病的进展和预防。

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.