High-throughput mapping of human antibody sequences to PfEMP1 malaria antigen specificity

项目摘要。单抗已被批准用于多种疾病的治疗。抗体 传染病的发展通常始于病原体特异性抗原(PSAg)的发现， 其次是动物模型中PSAg特异性抗体的升高。全人型PSAg特异性抗体 由来自临床免疫捐赠者的循环记忆B细胞编码的编码具有许多优点 在动物体内产生的抗体。不需要抗体人源化，它可以分离亲和力- 成熟的抗体与它们的天然目标抗原共同进化。在恶性疟原虫的情况下， 在这里研究的疟疾寄生虫，抗体的发现与几个重要的额外挑战有关。 抗原家族PfEMP1的成员已被确定为获得性保护性抗体的关键靶点- 对疟疾的媒介免疫，但充满了大量的克隆内和克隆间变异。这 极大地增加了对广泛交叉反应和中和抗体的鉴定的复杂性。此外，没有 研究PfEMP1特异性获得性免疫的实用动物模型(恶性疟原虫只感染人类)。 最后，到目前为止可用于询问特定于PfEMP1的功能的低吞吐量技术 抗体严重阻碍了对广泛反应的中和抗体的识别。人类 因此，在疟疾抗原发现中，单抗(HuMabs)在很大程度上仍然是一种未被探索的工具。 为了应对这些挑战，我们建议探索一种新技术的效用，以发现 编码PSAg特异性抗体的BCR序列(特异性目标1)，并评估交叉反应性和 这类抗体的体外功能(特定目标2)。具体地说，我们将应用最近描述的 Libra-seq技术，能够高通量地将抗体序列映射到抗原特异性， 使用一种特定类型的PfEMP1(VAR2CSA)，它与特定和 恶性疟原虫感染的严重并发症称为胎盘性疟疾。相应地，VAR2CSA特定于 抗体已被确定为胎盘疟疾自然获得保护的关键机制。 随后，我们将用经鉴定的序列生成针对VAR2CSA的重组huMabs Libra-seq来验证它们的交叉反应性，并在一定范围内评估重组抗体的功能 试管化验。我们之所以将最初的研究重点放在胎盘性疟疾上，是因为 胎盘疟疾获得性保护性免疫靶抗原(VAR2CSA型PfEMP1)良好 已经确定，因为胎盘疟疾是疟疾相关死亡和严重发病率的主要原因 在恶性疟原虫传播地区的孕妇及其后代中。 这些初步的努力将展示我们提出的方法的实用性，以识别广泛的 中和PfEMP1特异性抗体。因此，它们将为抗体发现工作提供一个框架。 在其他严重形式的恶性疟中，例如脑型疟疾，将产生广泛的影响。

Project Summary. Monoclonal antibodies are approved as therapeutics for a number of diseases. Antibody development in the infectious disease setting typically starts with pathogen-specific antigen (PSAg) discovery, followed by raising of PSAg-specific antibodies in animal models. Fully human, PSAg-specific antibodies encoded by circulating memory B cells from clinically immune donors present many advantages over antibodies raised in animals. There is no need for antibody humanization and it enables isolation of affinity- matured antibodies co-evolved with their native target antigens. In the case of Plasmodium falciparum, the malaria parasite studied here, antibody discovery is associated with several important additional challenges. Members of the antigen family PfEMP1 have been identified as key targets of acquired protective antibody- mediated immunity to malaria, but are fraught with substantial intra-clonal and inter-clonal variation. This greatly complicates identification of broadly cross-reactive and neutralizing antibodies. Furthermore, there is no practical animal model for the study of PfEMP1-specific acquired immunity (P. falciparum only infects humans). Finally, the low-throughput technologies so far available for interrogating the functionality of PfEMP1-specific antibodies have severely hampered the identification of broadly reactive, neutralizing antibodies. Human monoclonal antibodies (huMabs) therefore remain a largely unexplored tool in malaria antigen discovery. To address these challenges, we propose to explore the utility of a new technology for the discovery of BCR sequences encoding PSAg-specific antibodies (Specific Aim 1) and to assess the cross-reactivity and functionality of such antibodies in vitro (Specific Aim 2). Specifically, we will apply the recently described LIBRA-seq technology, which enables high-throughput mapping of antibody sequence to antigen specificity, using a specific type of PfEMP1 (VAR2CSA) that is centrally involved in the pathogenesis of a specific and severe complication to P. falciparum infection, called placental malaria. Correspondingly, VAR2CSA-specific antibodies have been identified as the key mechanism of naturally acquired protection from placental malaria. Subsequently, we will generate VAR2CSA-specific recombinant huMabs with the sequences identified by LIBRA-seq to verify their cross-reactivity and assess the functionality of the recombinant antibodies in a range of in vitro assays. The reason that we focus the initial studies proposed here on placental malaria is that the target antigen of acquired protective immunity to placental malaria (VAR2CSA-type PfEMP1) is well established, and because placental malaria is a major cause of malaria-related mortality and severe morbidity among pregnant women and their offspring in areas where transmission of P. falciparum occurs. These initial efforts will show the utility of our proposed approaches for identification of broadly neutralizing PfEMP1-specific antibodies. They will therefore provide a framework for antibody discovery efforts in other severe forms of P. falciparum malaria, e.g., cerebral malaria, and therefore will have a broad impact.