Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.
Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.
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DOI：
10.1371/journal.ppat.1010924
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发表时间：
2022-11
期刊：
影响因子：
6.7
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中科院分区：
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--
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妊娠期疟疾是由感染恶性疟原虫寄生虫引起的一个主要的全球性健康问题。严重的影响来自胎盘中受感染的红细胞的积累。在这里，被晚期血液阶段寄生虫感染的红细胞通过VAR2CSA型恶性疟原虫红细胞膜蛋白1（PfEMP 1）粘附蛋白粘附到胎盘硫酸软骨素A（CS）上。对胎盘疟疾的免疫是通过暴露获得的，并通过对VAR2CSA的抗体介导。通过进化，VAR2CSA蛋白在序列上多样化以逃避免疫识别，但保留其整体大分子结构以维持CS结合亲和力。这种结构保守性也可能使免疫女性产生广泛反应性的VAR2CSA抗体。在这里，我们显示了唯一已知的广泛反应性人单克隆抗体PAM 1.4与VAR2CSA复合的阴性染色和冷冻电镜结构。数据显示PAM 1.4的广泛VAR2CSA反应性如何通过与不同亚结构域的多个保守残基相互作用来实现，所述亚结构域形成远离VAR2CSA核心结构上的CS结合位点的构象表位。因此，虽然PAM 1.4可能代表一类通过诱导吞噬作用或NK细胞介导的细胞毒性介导胎盘疟疾免疫的抗体，但很可能广泛地CS结合抑制性抗体靶向CS结合位点处的其他表位。对这两种类型的广泛反应性单克隆抗体的了解可能有助于开发针对胎盘疟疾的疫苗。胎盘疟疾仍然是一个主要的全球健康问题。这种疾病是由疟原虫感染的红细胞在胎盘中积累引起的。寄生虫将多态性VAR2CSA蛋白家族的成员输出到红细胞表面以结合胎盘硫酸软骨素A（CS）。VAR2CSA蛋白家族在序列上多样化以避免免疫识别，但最近的VAR2CSA的CryoEM结构显示，所有变体折叠成由多个交织的“DBL”结构域组成的大的、致密的和稳定的核心，以及柔性的双DBL结构域尾部。对胎盘疟疾的免疫力是通过暴露获得的，并被认为是由广泛反应性抗体介导的，这些抗体通过中和CS结合或调理受感染的红细胞以进行细胞介导的杀伤而起作用。在这里，我们使用阴性染色和冷冻EM来解析针对VAR2CSA的广泛反应性抗体（PAM 1.4）的第一分子结构。我们发现，抗体结合高度保守的构象表位的VAR2CSA核心结构，远离先前确定的CS结合位点。这与新的数据一致，显示与CSA结合的寄生虫的PAM 1.4中和可忽略不计。作为充分表征的非中和性单克隆抗体，PAM 1.4及其Fc修饰现在可用于未来研究中的参考和基准，所述研究旨在在持续寻求针对胎盘疟疾的疫苗或治疗剂中定位、鉴定和鉴定针对VAR2CSA的功能性抗体。
Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4’s broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria. Placental malaria remains a major global health problem. The disease is caused by the accumulation of malaria parasite-infected red blood cells in the placenta. The parasites export members of the polymorphic VAR2CSA protein family onto the red blood cell surface to bind placental chondroitin sulphate A (CS). The VAR2CSA protein family have diversified in sequence to avoid immune recognition, but recent CryoEM structures of VAR2CSA shows that all variants fold into a large, dense and stable core comprised of multiple interwoven “DBL” domains, and a flexible two-DBL domain tail. Immunity to placental malaria is acquired through exposure and thought to be mediated by broadly reactive antibodies acting through neutralizing CS binding or opsonizing infected red blood cells for cell-mediated killing. Here, we used negative stain and cryo-EM to resolve the first molecular structure of a broadly reactive antibody (PAM1.4) against VAR2CSA. We find that the antibody binds a highly conserved conformational epitope on the VAR2CSA core structure, distant from previously determined CS binding sites. This aligns with new data showing negligible PAM1.4 neutralization of parasite binding to CSA. As a well-characterized non-neutralizing monoclonal antibody, PAM1.4 and Fc-modifications thereof, can now be used for reference and benchmarking in future studies aiming to map, identify and qualify functional antibodies against VAR2CSA in the continued quest for vaccines or therapeutics against placental malaria.
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