Potent neutralization of sars-cov-2 by hetero-bivalent alpaca nanobodies targeting the spike receptor-binding domain

Huan Ma, Weihong Zeng, Xiangzhi Meng, Xiaoxue Huang, Yunru Yang, Dan Zhao, Peigen Zhou, Xiaofang Wang, Changcheng Zhao, Yong Sun, Peihui Wang, Huichao Ou, Xiaowen Hu, Yan Xiang, Tengchuan Jin

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


Cellular entry of SARS-CoV-2 requires the binding between the receptorbinding domain (RBD) of the viral Spike protein and the cellular angiotensin-converting enzyme 2 (ACE2). As such, RBD has become the major target for vaccine development, whereas RBD-specific antibodies are pursued as therapeutics. Here, we report the development and characterization of SARS-CoV-2 RBD-specific VHH/nanobody (Nb) from immunized alpacas. Seven RBD-specific Nbs with high stability were identified using phage display. They bind to SARS-CoV-2 RBD with affinity KD ranging from 2.6 to 113nM, and six of them can block RBD-ACE2 interaction. The fusion of the Nbs with IgG1 Fc resulted in homodimers with greatly improved RBD-binding affinities (KD ranging from 72.7 pM to 4.5nM) and nanomolar RBD-ACE2 blocking abilities. Furthermore, the fusion of two Nbs with nonoverlapping epitopes resulted in hetero-bivalent Nbs, namely, aRBD-2-5 and aRBD-2-7, with significantly higher RBD binding affinities (KD of 59.2 pM and 0.25nM) and greatly enhanced SARS-CoV-2 neutralizing potency. The 50% neutralization doses of aRBD-2-5 and aRBD-2-7 were 1.22 ng/ml (∼0.043 nM) and 3.18 ng/ml (∼0.111nM), respectively. These high-affinity SARS-CoV-2 blocking Nbs could be further developed into therapeutics, as well as diagnostic reagents for COVID-19.

Original languageEnglish (US)
Article numbere02438-20
JournalJournal of virology
Issue number10
StatePublished - May 2021


  • Alpaca
  • Antibody
  • COVID-19
  • Hetero-bivalent
  • Nanobody
  • SARS-CoV-2

ASJC Scopus subject areas

  • Insect Science
  • Virology
  • Microbiology
  • Immunology


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