Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.

恶性疟原虫（Plasmodium falciparum）感染的红细胞（pRBC，parasitized red blood cell）与正常红细胞形成玫瑰花结（rosettes）的能力，与疟原虫的毒力直接相关；而能够削弱玫瑰花结形成的红细胞多态性，可对重型疟疾产生保护作用。黏附素PfEMP1（恶性疟原虫红细胞膜蛋白1，Plasmodium falciparum Erythrocyte Membrane Protein 1）介导该结合过程，特异性抗体可阻止感染红细胞在微血管内的扣押，这一现象已在动物模型中得到证实。本研究证实，可破坏玫瑰花结的抗体所靶向的表位（epitope），汇聚于PfEMP1-DBL1α的h6与h7 α螺旋之间、连接二者的3号亚结构域（SD3，subdomain 3）的环区。结合SD3环区表位的单克隆抗体（monoclonal antibody）与多克隆IgG（免疫球蛋白G，immunoglobulin G），均可染色感染红细胞的表面、破坏玫瑰花结，并阻断重组NTS-DBL1α与正常红细胞的直接结合。将针对NTS-DBL1α制备的多克隆IgG通过SD3环肽进行抗体耗竭后，其抗玫瑰花结活性会被完全消除。使用重组1号亚结构域（SD1，subdomain 1）、2号亚结构域（SD2，subdomain 2）或SD3进行免疫，均可产生可结合感染红细胞表面的抗体，但仅SD3免疫组动物的血清能够破坏玫瑰花结。系统发育分析显示，SD3序列可分为两个类群（A/B组）：A组包含与SD2结构域中两个半胱氨酸残基相关的玫瑰花结相关序列，而B组则包含带有三个及以上半胱氨酸残基的序列。本研究结果表明，PfEMP1-DBL1α的SD3环区是抗玫瑰花结活性的重要靶点，阐明了变异株特异性抗玫瑰花结抗体产生的分子基础。