Portero Carolina E, Smith Claire, Zhou Yuxi, Marchán-Rivadeneira M Raquel, Wu Shiyong, Han Yong
Department of Chemistry and Biochemistry, Ohio University, Athens, OH, 45701, USA.
Edison Biotechnology Institute, Ohio University, Athens, OH, 45701, USA.
Probiotics Antimicrob Proteins. 2025 Sep 11. doi: 10.1007/s12602-025-10758-1.
SARS-CoV-2 viral infection can be inhibited by blocking the interaction between the viral spike protein and the human receptor angiotensin-converting enzyme 2 (hACE2). The development of specific spike inhibitors using nanobodies, the antigen-binding region of llamas' antibodies, arose as a promising therapeutic method against SARS-CoV-2. However, one limitation of nanobodies is that they cannot be used directly in the human body due to their susceptibility to degradation. Bacteria-based delivery systems provide site-specific targeted action that can circumvent nanobody degradation. Here, we report the development of a genetically modified bacterium expressing anti-SARS-CoV-2 nanobodies that can inhibit the interaction between the hACE2 receptor and the receptor-binding domain (RBD) of the spike protein. Lactococcus lactis, a human symbiont probiotic bacterium, was selected to express nanobodies attached to their cell surface. Our data shows that FLAG-tagged anti-SARS-CoV-2 nanobodies were detected on the cell surface of recombinant L. lactis strains by flow cytometry and immunofluorescence without permeabilization. Furthermore, nanobodies are functional and can bind the RBD region from the spike protein in a dose-dependent manner. Inhibition of the hACE2-RBD interaction in cellular assays was quantified using a pseudotype lentivirus that mimics SARS-CoV-2 in an adaptation of the neutralization assay. Our results suggested that the recombinant bacteria can inhibit viral infectivity in more than 50% compared with a control without bacteria in a neutralization assay. These outcomes suggest that the engineered strain can be used in the future as a new therapeutic tool in COVID-19 prevention.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒感染可通过阻断病毒刺突蛋白与人受体血管紧张素转换酶2（hACE2）之间的相互作用来抑制。利用纳米抗体（骆驼抗体的抗原结合区域）开发特异性刺突抑制剂，成为一种有前景的抗SARS-CoV-2治疗方法。然而，纳米抗体的一个局限性是，由于它们易降解，不能直接在人体中使用。基于细菌的递送系统提供位点特异性靶向作用，可避免纳米抗体降解。在此，我们报告了一种表达抗SARS-CoV-2纳米抗体的基因工程细菌的开发，该纳米抗体可抑制hACE2受体与刺突蛋白的受体结合域（RBD）之间的相互作用。乳酸乳球菌是一种人类共生益生菌，被选来表达附着在其细胞表面的纳米抗体。我们的数据表明，通过流式细胞术和免疫荧光在未通透处理的情况下，在重组乳酸乳球菌菌株细胞表面检测到了带有FLAG标签的抗SARS-CoV-2纳米抗体。此外，纳米抗体具有功能，能以剂量依赖方式结合刺突蛋白的RBD区域。在细胞试验中，使用一种模拟SARS-CoV-2的假型慢病毒，通过中和试验的改进方法来定量hACE2-RBD相互作用的抑制情况。我们的结果表明，在中和试验中，与无细菌的对照相比，重组细菌可将病毒感染性抑制50%以上。这些结果表明，工程菌株未来可作为预防2019冠状病毒病的一种新治疗工具。
