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

项目摘要。单克隆抗体被批准作为许多疾病的治疗剂。抗体 感染性疾病的发展通常始于病原体特异性抗原（PSAg）的发现， 随后在动物模型中产生PSA g特异性抗体。全人PSA g特异性抗体 由来自临床免疫供体的循环记忆B细胞编码的免疫调节剂具有许多优点， 动物体内产生的抗体不需要抗体人源化，并且它能够分离亲和性- 成熟抗体与它们的天然靶抗原共进化。在恶性疟原虫的情况下， 在这里研究疟疾寄生虫时，抗体的发现与几个重要的额外挑战有关。 抗原家族PfEMP 1的成员已被鉴定为获得性保护性抗体的关键靶点， 介导的疟疾免疫，但充满了大量的克隆内和克隆间的变化。这 使广泛交叉反应性和中和性抗体的鉴定变得非常复杂。此外，没有 用于研究PfEMP 1特异性获得性免疫的实用动物模型（恶性疟原虫仅感染人类）。 最后，目前可用于询问PfEMP 1特定的功能的低吞吐量技术 抗体严重阻碍了广泛反应性中和抗体的鉴定。人类 因此，单克隆抗体（huMab）在疟疾抗原发现中仍然是一种很大程度上未开发的工具。 为了应对这些挑战，我们建议探索一种新技术的实用性， 编码PSA g特异性抗体（特异性Aim 1）的BCR序列，并评估交叉反应性， 在体外研究这些抗体的功能性（具体目标2）。具体来说，我们将应用最近描述的 LIBRA-seq技术，能够将抗体序列高通量映射到抗原特异性， 使用一种特定类型的PfEMP 1（VAR 2CSA），它主要参与一种特定的和 恶性疟原虫感染的严重并发症，称为胎盘疟疾。相应地，VAR 2CSA特异性 抗体已被确定为自然获得的保护免受胎盘疟疾的关键机制。 随后，我们将产生VAR 2CSA特异性重组huMab，其序列由 LIBRA-seq，以验证其交叉反应性并评估重组抗体在一定范围内的功能性。 的体外试验。我们把这里提出的初步研究集中在胎盘疟疾上的原因是， 胎盘疟疾获得性保护性免疫的靶抗原（VAR 2CSA型PfEMP 1）是良好的 由于胎盘疟疾是疟疾相关死亡和严重发病的主要原因， 在发生恶性疟原虫传播地区的孕妇及其后代中。 这些初步的努力将显示我们提出的方法的实用性，广泛的识别 中和PfEMP 1特异性抗体。因此，它们将为抗体发现工作提供框架 在恶性疟原虫疟疾的其他严重形式中，例如，脑型疟疾，因此将产生广泛的影响。

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.