Institute of Anatomy, University of Bern, 3012, Bern, Switzerland.
Labor Spiez, Federal Office for Civil Protection, 3700, Spiez, Switzerland.
Sci Rep. 2023 Apr 4;13(1):5537. doi: 10.1038/s41598-023-32269-1.
Clinical studies have proven antiviral effectiveness of treatment with a Designed Ankyrin Repeat Protein (DARPin) specific against the spike protein of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). More information on transport mechanisms and efficiency to the site of action is desirable. Transepithelial migration through air-liquid interface (ALI) cultures of reconstituted human bronchial epithelia (HBE) was assessed by Enzyme-Linked Immunosorbent Assays and Confocal Laser Scanning Microscopy for different DARPin designs in comparison to a monoclonal antibody. Antiviral efficacy against authentic SARS-CoV-2, applied apically on HBE, was investigated based on viral titers and genome equivalents, after administration of therapeutic candidates on the basal side. Transepithelial translocation of all DARPin candidates and the monoclonal antibody was efficient and dose dependent. Small DARPins and the antibody migrated more efficiently than larger molecules, indicating different transport mechanisms involved. Microscopic analyses support this, demonstrating passive paracellular transport of smaller DARPins and transcellular migration of the larger molecules. All therapeutic candidates applied to the basal side of HBE conferred effective protection against SARS-CoV-2 infection. In summary, we have shown that DARPins specific against SARS-CoV-2 translocate across intact airway epithelia and confer effective protection against infection and viral replication.
临床研究已经证明了针对严重急性呼吸综合征冠状病毒 2 型（SARS-CoV-2）刺突蛋白的设计锚蛋白重复蛋白（DARPin）治疗的抗病毒效果。更希望了解其向作用部位的转运机制和效率。通过重建的人支气管上皮（HBE）的气液界面（ALI）培养物中的酶联免疫吸附测定和共聚焦激光扫描显微镜，评估了不同 DARPin 设计与单克隆抗体相比在穿过上皮细胞的迁移能力。针对 HBE 表面的真实 SARS-CoV-2，在基底侧给予治疗候选物后，基于病毒滴度和基因组当量，研究了针对 HBE 表面的抗病毒功效。所有 DARPin 候选物和单克隆抗体的跨上皮转运均有效且呈剂量依赖性。小分子 DARPins 和抗体的迁移效率高于大分子，表明涉及不同的转运机制。显微镜分析支持这一点，表明较小的 DARPins 通过被动的细胞旁途径转运，而较大的分子通过细胞内途径迁移。所有应用于 HBE 基底侧的治疗候选物均能有效防止 SARS-CoV-2 感染。总之，我们已经表明，针对 SARS-CoV-2 的 DARPin 可以穿过完整的气道上皮细胞，并有效防止感染和病毒复制。
