A Competitive Panning Method Reveals an Anti-SARS-CoV-2 Nanobody Specific for an RBD-ACE2 Binding Site

Abstract

Most neutralizing antibodies neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by directly blocking the interactions between the spike glycoprotein receptor-binding domain (RBD) and its receptor, human angiotensin-converting enzyme 2 (ACE2). Here, we report a novel nanobody (Nb) identified by an RBD-ACE2 competitive panning method that could specifically bind to the RBD of SARS-CoV-2 with a high affinity (EC50 = 0.03 nM) and successfully block the binding between the RBD and ACE2 recombinant protein. A structural simulation of the RBD-VHH complex also supports a mechanism of the Nb to block the interaction between the RBD and ACE2. A pseudovirus assay of the Nb showed it could neutralize the WT pseudovirus with high potency (IC50 = 0.026 μg/mL). Furthermore, we measured its binding to phages displaying RBDs of different SARS-CoV-2 variants and found that it could bind to recombinant phages displaying the RBD of beta and delta variants. This study also provides a method of phage library competitive panning, which could be useful for directly screening high-affinity antibodies targeting important functional regions.

Dendritic Cell‐Mediated Cross‐Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS‐CoV‐2

“…RBD-targeted V HH s have shown effective in neutralizing SARS-CoV-2 infection in vitro and in protecting animals from SARS-CoV-2 challenge, [23,25,27,28,54] making them a valuable alternative for the development of multivalent and multiparatopic agents (targeting multiple viral epitopes) with improved neutralization efficiency and decreased vulnerability to escape mutations. [29,55,56] Here, we used the high-affinity biparatopic V HH -tandem, TN, previously described to potently neutralize SARS-CoV-2 strain Wuhan-Hu-1 and prevent the emergence of escape mutants.…”

Revolutionizing antiviral therapy with nanobodies: Generation and prospects

“…This study [36] demonstrate how Nbs can be used to target specific proteins on the surface of viral hosts, such as human cells infected with SARS-CoV-2. By blocking these proteins, Nbs can prevent viruses from entering or replicating within host cells.…”

“…developed Nb that could bind to angiotensin I-converting enzyme-2 (ACE2) protein. By binding to ACE2, these Nbs prevented the virus from infecting cells in laboratory experiments [36] .…”

Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering

“…Additionally, 2 CV elution at 0.5 mL/min was completed at an imidazole gradient of 20-500 mM. The eluted fractions were collected and analyzed on 12% SDS-PAGE [18,19]. The samples having the band of protein with a corresponding size to that of NP 1-419 , NP 121-419 , and NP 250-365 were pooled down, respectively, and buffer was exchanged with 10 mM sodium phosphate buffer pH 8 and 150 mM NaCl buffer.…”