Müller Luisa, Dabbiru Venkata A S, Rutten Lucy, Bos Rinke, Zahn Roland, Handtke Stefan, Thiele Thomas, Palicio Marta, Esteban Olga, Broto Marta, Gordon Tom Paul, Greinacher Andreas, Wang Jing Jing, Schönborn Linda
Institut für Transfusionsmedizin, Universitätsmedizin Greifswald, 17489 Greifswald, Germany.
Janssen Vaccines & Prevention BV, 2333 CN Leiden, The Netherlands.
Vaccines (Basel). 2024 Dec 24;13(1):3. doi: 10.3390/vaccines13010003.
BACKGROUND/OBJECTIVES: Adenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant antibodies (rAbs) derived from VITT patient characteristic amino acid sequences of anti-PF4 IgG are an alternative to study VITT pathophysiology.
Amino acid sequences of the variable region of immunoglobulin light and heavy chain of anti-PF4 IgG derived from VITT patients were obtained by mass spectrometry sequencing and rAbs were synthetized by reverse-engineering. Six different rAbs were produced: CR23003, CR23004, and CR23005 (from a patient vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, and CR22050 and CR22066 (from two different patients vaccinated with Vaxzevria, AstraZeneca (Cambridge, UK)). These rAbs were further characterized using anti-PF4 and anti-PF4/heparin IgG ELISAs, rapid anti-PF4 and anti-PF4/polyanion chemiluminescence assays, and PF4-induced platelet activation assay (PIPA) and their capacity to induce procoagulant platelets.
rAbs bound to PF4 alone, but not to PF4/polyanion complexes in rapid chemiluminescence assays. Chemiluminescence assays and both anti-PF4 IgG and anti-PF4 IgG/heparin ELISA showed concentration-dependent PF4 binding of all six rAbs, however, with different reactivities among them. PIPA showed a similar, concentration-dependent platelet activation pattern. rAbs varied in their reactivity and the majority of the tested rAbs were able to induce procoagulant platelets.
The six rAbs derived from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these rAbs offer an attractive new option to study VITT pathophysiology.
背景/目的：基于腺病毒载体的新冠疫苗极少引发疫苗诱导的免疫性血小板减少症和血栓形成（VITT），这是一种由抗血小板因子4（PF4）的IgG抗体引起的严重不良反应。为研究VITT，患者样本至关重要，但已成为稀缺资源。源自VITT患者抗PF4 IgG特征性氨基酸序列的重组抗体（rAb）是研究VITT病理生理学的一种替代方法。
通过质谱测序获得源自VITT患者的抗PF4 IgG免疫球蛋白轻链和重链可变区的氨基酸序列，并通过逆向工程合成rAb。制备了六种不同的rAb：CR23003、CR23004和CR23005（来自接种Jcovden的患者，强生公司 - 杨森制药（比利时贝尔瑟）），CR22046、CR22050和CR22066（来自接种Vaxzevria的两名不同患者，阿斯利康公司（英国剑桥））。使用抗PF4和抗PF4/肝素IgG ELISA、快速抗PF4和抗PF4/聚阴离子化学发光测定法以及PF4诱导的血小板活化测定法（PIPA）对这些rAb进行进一步表征，并检测它们诱导促凝血血小板的能力。
在快速化学发光测定中，rAb仅与PF4结合，而不与PF4/聚阴离子复合物结合。化学发光测定以及抗PF4 IgG和抗PF4 IgG/肝素ELISA均显示所有六种rAb与PF4的结合呈浓度依赖性，然而，它们之间的反应性不同。PIPA显示出类似的、浓度依赖性的血小板活化模式。rAb的反应性各不相同，大多数测试的rAb能够诱导促凝血血小板。
源自VITT患者的六种rAb反映了VITT典型的结合能力和激活血小板的能力。因此，这些rAb为研究VITT病理生理学提供了一个有吸引力的新选择。
