Dissecting Polyclonal Sera to Reveal Correlates of Productive Immune Responses to HIV

项目总结/摘要 在慢性HIV感染期间出现的广泛中和抗体（bnAb）突出了 人类免疫系统进化出能够识别高度可变和糖基化的抗体的能力 proteins.然而，通过初免-加强疫苗引发这些类型的抗体仍然是一个困难的挑战。 最近，基于结构的合理疫苗设计策略重新激发了对有效HIV的追求， 疫苗这种方法依赖于对鞋底与鞋底之间的相互作用的高分辨率理解。 HIV表面上的中和靶标、包膜糖蛋白（Env）和可用于工程化的bnAb 免疫原，旨在重现幼稚个体中的bnAb应答。这种方法是迭代的， 依赖于表征动物对这些免疫原的免疫应答的能力， 免疫原设计血清中和测定和ELISA结合提供了对引起的免疫应答的估计。 免疫反应，但不是合理设计所需的分子细节。通过丙氨酸的表位作图 扫描、抗原特异性单克隆抗体分离和下一代测序提供了进一步的 细节，但每一个都有局限性，并且是非常耗时的工作。因此，我们制定了 一种新的方法来解剖多克隆免疫反应，以提供一个完整的和定量的地图， 靶向的表位是快速的，并且可以真实的时间通知加强策略。通过净化免疫复合物 在Env三聚体免疫原和来自免疫动物的血清抗体之间形成，并通过 在电子显微镜下，我们直接观察多克隆抗体反应。这些反应可以量化 直接或结合深度测序和质谱法。我们已经证明了概念， 用BG 505 SOSIP.664天然样Env三聚体免疫家兔，并建议使用我们的方法学 最近完成的一项大型非人灵长类动物（NHP）疫苗接种研究（138只动物）， 使用BG 505 SOSIP.664平台的各种免疫策略。在这项研究中， 有或没有产生有效的自体中和抗体反应，以及产生 一定程度的中和宽度因此，我们有一个独特的机会来了解分子 这种免疫应答的基础，并描绘多克隆抗体中的表型特征。 抗体反应，可用于指导艾滋病毒疫苗的开发。此外，我们的研究可以应用于 664免疫原用于即将进行的人体临床试验的人血清样品。 因此，我们可以生成人类和NHP之间的比较数据，以提高临床前 动物模型仍然是迭代合理的HIV疫苗设计过程的重要组成部分。

Project Summary/Abstract Broadly neutralizing antibodies (bnAbs) that arise during chronic HIV infection highlight the remarkable capacity of the human immune system to evolve antibodies that can recognize highly variable and glycosylated proteins. Eliciting these types of antibodies via a prime-boost vaccine however remains a difficult challenge. Recently, structure-based rational vaccine design strategies have reinvigorated the pursuit of an effective HIV vaccine. This approach relies on a high-resolution understanding of the interaction between the sole neutralizing target on the surface of HIV, envelope glycoprotein (Env), and bnAbs that can be used to engineer immunogens designed to recapitulate bnAb responses in naïve individuals. This approach is iterative and relies on the ability to characterize immune responses in animals to such immunogens to refine the immunogen design. Serum neutralization assays and ELISA binding provide an estimation of the elicited immune response but not the molecular details required for rational design. Epitope mapping via alanine scanning, antigen specific monoclonal antibody isolation, and next generation sequencing provide further details, but each has limitations and are very time-consuming endeavors. We have therefore developed a novel method to dissect polyclonal immune responses to provide a complete and quantitative map of the epitopes targeted that is rapid and can inform boosting strategies in real time. By purifying immune complexes formed between Env trimer immunogens and serum antibodies from immunized animals, and imaging them by electron microscopy, we directly visualize polyclonal antibody responses. These responses can be quantitated directly or in combination with deep sequencing and mass spectroscopy. We have shown proof of concept in rabbits immunized with BG505 SOSIP.664 native-like Env trimers, and propose to use our methodology to study a recently completed large non-human primate (NHP) vaccination study (138 animals) that tested various immunization strategies using the BG505 SOSIP.664 platform. Within this study there are animals that did or did not develop potent autologous neutralizing antibody responses, as well as animals that developed some level of neutralization breadth. Hence, we have a unique opportunity to understand the molecular underpinnings of such immune responses, and to delineate phenotypic signatures within the polyclonal antibody responses that can be used to guide HIV vaccine development. Further, our studies can be applied to human serum samples for an upcoming human clinical trial using the same BG505 SOSIP.664 immunogen. And thus, we can generate comparative data between humans and NHPs to improve the utility of pre-clinical animal models that remain an essential component of the iterative rational HIV vaccine design process.