New Technology for High-Resolution Antibody Profiling for SARS-CoV-2

摘要/摘要 到目前为止，仅在美国，SARS-CoV-2病毒就已经感染了3500万人，导致60多万人死亡。尽管 疫苗的初步成功，越来越多传染性变种的出现，如Delta变种， 再加上疫苗的犹豫不决和不够有效的治疗，导致了一种持续和深化的 全国性和世界性的大流行危机。此外，相当一部分(~35%)的新冠肺炎患者，即使是那些 最初没有症状，遭受长期衰弱影响的人(“长期冠状病毒病”)。最近的报告表明， SARS-CoV-2诱导的具有潜在致死性炎症反应的特异性抗体的结构 新冠肺炎。研究还将抗体反应与长期冠状病毒感染联系起来。此外，抗体的性质 对疫苗接种的反应与突破性感染相关。因此，快速执行高性能- 分辨率，高度多元化的抗体反应图谱可以提供一个重要的工具，最终导致更多 对急性和长期疾病进行有效的诊断、预测、疫苗和治疗。但是，当前 抗体分析方法产生了对体液曲目的非常有限的看法。为了解决这一未得到满足的需求， AmberGen计划在第二阶段进一步开发其质谱珠阵技术，以深入 免疫分析，称为PC-BAMS-IP™。这将为研究人员提供一种新的强大的工具来研究高 与当前技术不同的是，分辨率抗体图谱促进了2维的多路复用。这是 利用带有病毒抗原的PC-MT编码小球结合血清完成 抗体，以及一系列PC-MT编码的探针，可同时测量结合珠子的全宽度 抗体类型。珠子阵列的质谱学成像(MSI)有助于解码数千个 不同的PC-MT，从而揭示了抗体反应的全部复杂性，并提供了一种将其与 疾病严重程度/结果。以SARS-CoV-2为重点的可行性研究证实了PC-BAMS-IP™的能力 同时对Fab特性(抗原结合功能)和Fc进行二维抗体分析 特征(免疫效应功能)。拟议的为期两年的第二阶段项目将扩大这一进展，包括：一) 25种SARS-CoV-2即插即用PC-MT编码珠的设计、合成及评价 固定化和25个PC-MT探针同时查询结合珠血清的一系列Fc特性 抗体；ii)使用对照和新冠肺炎恢复期血清对PC-BAMS-IP™分析的初步验证， 包括与Luminex®xMAP®技术的比较，Luminex®xMAP®技术是一维多路复用的现有黄金标准 抗体谱分析；iii)证明PC-BAMS-IP™可以区分重度和轻度新冠肺炎。这部作品 我们将通过与包括凯西·科斯特洛教授在内的领先专家的持续合作来促进这一进程。 著名质谱学专家)，Jason Amsden博士(杜克大学，质谱仪 发展)，拉姆·古姆穆鲁教授(波士顿大学，微生物学副主席，领先的病毒学家)和普拉门教授 伊万诺夫(BU，凯克网络生理学实验室主任，高级统计物理)。

Summary/Abstract The SARS-CoV-2 virus has infected to date 35 million and killed over 600,000 persons in the U.S. alone. Despite initial success of the vaccines, the emergence of increasingly more infectious variants such as the delta variant, coupled with vaccine hesitancy and insufficiently effective therapies, has resulted in a continued and deepening national and world-wide pandemic crisis. Moreover, a significant fraction of COVID-19 patients (~35%), even those who are initially asymptomatic, suffer long-term debilitating effects (“long-COVID”). Recent reports correlate the structure of specific SARS-CoV-2 induced antibodies with potentially lethal proinflammatory responses in acute COVID-19. Studies have also linked the antibody response to long-COVID. Moreover, the nature of the antibody response to vaccination correlates with breakthrough infections. Thus, the ability to rapidly perform high- resolution, highly multiplexed antibody response profiling can provide an essential tool, ultimately leading to more effective diagnostics, prognostics, vaccines and treatments for both acute and long-term disease. However, current antibody profiling methods produce a very limited view of the humoral repertoire. To address this unmet need, AmberGen proposes to further develop in Phase II its mass spectrometric bead-array technology for in-depth immune-profiling, termed PC-BAMS-IP™. This will provide researchers with a new and powerful tool for high- resolution antibody profiling which unlike current technology facilitates 2-dimensions of multiplexing. This is accomplished using arrayed photocleavable mass-tag (PC-MT) encoded beads bearing viral antigens to bind serum antibodies, along with a range of PC-MT encoded probes to simultaneously measure the full breadth of bead-bound antibody types. Mass spectrometry imaging (MSI) of the bead-arrays facilitates the decoding of thousands of different PC-MTs, thereby revealing the full complexity of the antibody response and a means to correlate it with disease severity/outcome. Feasibility studies focused on SARS-CoV-2 demonstrate the ability of PC-BAMS-IP™ to perform simultaneous 2-dimensional antibody profiling of both the Fab traits (antigen binding function) and Fc traits (immune effector function). The proposed 2-year Phase II project will expand on this progress, including: i) design, synthesis and evaluation of 25 plug-and-play PC-MT encoded beads for SARS-CoV-2 antigen immobilization and 25 PC-MT probes to simultaneously query a range of Fc traits of the bead-bound serum antibodies; ii) initial validation of the PC-BAMS-IP™ assay using control and COVID-19 convalescent sera, including comparison to Luminex® xMAP® technology, the existing gold-standard for 1-dimensional multiplex antibody profiling; and iii) demonstrate that PC-BAMS-IP™ can distinguish severe and mild COVID-19. This work will be facilitated by our continued collaboration with leading experts including Prof. Cathy Costello (BU, world- renowned mass spectrometry expert), Dr. Jason Amsden (Duke University, mass spectrometric instrument development), Prof. Rahm Gummuluru (BU, Vice Chair of Microbiology, a leading virologist), and Prof. Plamen Ivanov (BU, Director, Keck Laboratory for Network Physiology, advanced statistical physics).