ABSTRACT
SARS-CoV-2 and its variants, such as the Omicron continue to threaten public health. The virus recognizes the host cell by attaching its Spike (S) receptor-binding domain (RBD) to the host receptor, ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here, we report the isolation and biological and structural characterization of a single-chain antibody (nanobody) from RBD-immunized alpaca. The nanobody, named DL28, binds to RBD tightly with a K D of 1.56 nM and neutralizes the original SARS-CoV-2 strain with an IC50 of 0.41 µg mL-1. Neutralization assays with a panel of variants of concern (VOCs) reveal its wide-spectrum activity with IC50 values ranging from 0.35 to 1.66 µg mL-1 for the Alpha/Beta/Gamma/Delta and an IC50 of 0.66 µg mL-1 for the currently prevalent Omicron. Competition binding assays show that DL28 blocks ACE2-binding. However, structural characterizations and mutagenesis suggest that unlike most antibodies, the blockage by DL28 does not involve direct competition or steric hindrance. Rather, DL28 may use a "conformation competition" mechanism where it excludes ACE2 by keeping an RBD loop in a conformation incompatible with ACE2-binding.
ABSTRACT
SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 µg mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-Å resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.
Subject(s)
SARS-CoV-2 , Single-Domain Antibodies , Angiotensin-Converting Enzyme 2 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Protein Binding , Protein Engineering , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/chemistryABSTRACT
BACKGROUND: Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. RESULTS: Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral Nsps for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. CONCLUSIONS: Our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.