The emergence of new escape mutants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-angiotensin-converting enzyme 2 interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif of the spike protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, using mCherry fluorescence-based reporter assay, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid and efficient methodology to use peptide sequences from a protein–receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.

严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的新逃逸突变体的出现，加剧了其在人群中的渗透，并恢复了其全球大流行的地位。因此，在短时间内开发针对新出现的 SARS-CoV 变种和其他病毒的有效抗病毒疗法至关重要。通过破坏刺突糖蛋白-血管紧张素转换酶 2 相互作用来阻止 SARS-CoV-2 进入人类宿主细胞，已被用于疫苗开发和单克隆抗体治疗。与之前的报道不同，我们的研究使用来自刺突蛋白受体结合基序的九个氨基酸肽作为表位。我们报告了一种有效的纳米抗体 N1.2 的鉴定，它可以阻止含有假病毒的 SARS-CoV-2 刺突作为表面糖蛋白的进入。此外，使用基于 mCherry 荧光的报告基因检测，我们观察到二价版本的 N1.2 纳米抗体对 hCoV19 (Wuhan/WIV04/2019) 和 Omicron (BA.1) 假型刺突病毒具有更有效的中和作用。总之，我们的研究提出了一种快速有效的方法，使用蛋白质-受体相互作用界面的肽序列作为表位来筛选针对潜在致病靶点的纳米抗体。我们建议这种方法将来也可以广泛扩展到针对其他病毒和病原体。