The influence of evolutionary landscapes on protective antibody development

现代疫苗发展中最紧迫的问题之一是不良的异亚型中和 由对公众健康构成重大威胁的高度多样化的病毒引起的抗体反应。启发式 这些所谓的广谱中和抗体(BNAbs)--即结合和中和许多不同 病毒亚型-在人类患者中很少见，这使疫苗开发变得复杂，并允许重复 每年人类患者的病毒感染(如流感和登革热)或重叠感染(艾滋病毒)。在……里面 特别是，目前的疫苗诱导的bNAb效价不足，无法持久地预防目前的ALL 流行性和大流行性流感病毒株。因此，了解为什么选择某些抗体是至关重要的 (即为什么它们是免疫优势的)，这是一个与有效疫苗设计直接相关的问题 治疗许多顽固不化的传染病。 我们的长期目标是绘制抗体谱系的发展图，以便能够预测 可能的反应作为操纵抗体选择过程的工具。我们的主要目标是测试 假设抗流感bNAbs的抗体进化图景比亚型更有限- 这种差异解释了为什么这些bNAb在人类中通常不是高水平的选择。 为了验证这一假设，我们将在酵母上展示流感特异性抗体库，并使用变型 排序和深度测序流水线，以评估文库的每个成员变体的亲和力和 非特定绑定属性。然后，这些海量的功能数据将用于网络分析 为每个体细胞bNab和亚型特异性头部抗体重建合理的进化轨迹。这个 建议研究的动机是由于迫切需要开发方法来绘制和操纵地图 体内抗体亲和力成熟规则，以开发针对高度感兴趣的难治性病原体的疫苗 公共卫生，包括流感、登革热和艾滋病毒。 拟议的研究项目将通过追求三个具体目标来进行： 1)确定两种异亚型HA干细胞结合蛋白从生殖系到成熟人的个体发育； 2)确定从生殖系到成熟人类的四种异亚型和亚型抗体的个体发育 特定的HA头部粘结剂； 3)确定具有代表性的生殖系Ab的进化轨迹的数目。 这种方法是创新的，因为它结合了一个独特的假说和最先进的蛋白质 需要工程工具来评估假设，这一点很重要，因为这里生成的数据将 解释为什么bNab对HA免疫原的长期反应是如此罕见。在本文中提出的方法 应用还可以加速疫苗、预防和治疗药物的合理结构设计 对抗一系列人类病原体。

One of the most pressing issues in modern vaccine development is the poor heterosubtypic neutralizing antibody responses elicited by highly diverse viruses that pose major threats to public health. Elicitation of these so-called broadly neutralizing antibodies (bnAbs) – or antibodies that bind and neutralize many different viral subtypes – are rare in human patients, which complicates vaccine development and allows for repeated annual viral infections (e.g., Influenza and Dengue) or even superinfection (HIV) in human patients. In particular, current vaccines elicit bnAbs at insufficient titers for long-lasting protection against all currently circulating and pandemic Influenza strains. Therefore, it is critical to understand why some Abs are selected over others (i.e., why they are immunodominant), an issue directly relevant to the design of effective vaccines for many recalcitrant infectious diseases. Our long-term objective is to map the development of antibody lineages in a way that enables the prediction of likely responses as a tool to manipulate the process of antibody selection. Our primary objective is to test the hypothesis that the antibody evolutionary landscape is more limited for anti-Influenza bnAbs than for subtype- specific Abs, with this difference explaining why these bnAbs are not often selected at high levels in humans. To test this hypothesis we will display Influenza-specific antibody libraries on yeast and use a transformative sorting and deep sequencing pipeline to evaluate each member variant of the library for its affinity and nonspecific binding properties. This massive functional data will then be used for a network analysis to reconstruct plausible evolutionary trajectories for each somatic bnAb and subtype-specific head Ab. The motivation for the proposed research is guided by the urgent need for develop methods to map and manipulate rules of in vivo antibody affinity maturation to develop vaccines against refractory pathogens of high interest to public health including Influenza, Dengue, and HIV. The proposed research project will be carried out by pursuing three specific aims: 1) Determine ontogeny from germ line to mature human antibodies for two heterosubtypic HA stem binders; 2) Determine ontogeny from germ line to mature human antibodies for four heterosubtypic and subtype- specific HA head binders; 3) Determine the number of evolutionary trajectories from a representative germline Ab. This approach is innovative because it combines a unique hypothesis with state of the art protein engineering tools needed to evaluate the hypothesis, and it is significant because the data generated here will illuminate why long-lasting bnAb responses to HA immunogens are so rare. The approach raised in this application may also expedite rational structure-based design of vaccines, prophylactics, and therapeutics against a range of human pathogens.