Acquisition of a natural mutation primes SARS-CoV-2 for neutralization by a cross-reactive antibody

A team of scientists from the United States reveals that the acquisition of a natural mutation makes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptible to neutralization by a cross-reactive SARS-CoV antibody. The study is currently available on the bioRxiv* preprint server.

Since the emergence of the coronavirus disease 2019 (COVID-19) pandemic, many studies have been conducted to identify effective therapeutics or vaccines to contain the rapid spread of SARS-CoV-2. The spike protein on the surface of SARS-CoV-2 is considered the primary antigenic target for neutralizing antibodies. The receptor-binding domain (RBD) of the spike protein binds to host cell angiotensin-converting enzyme 2 (ACE2) to facilitate the viral entry into host cells. The amino acid sequence similarity between the spike protein of SARS-CoV-2 and SARS-CoV is 77%, justifying the difference in antigenicity and immunogenicity between these two coronaviruses.

In the case of SARS-CoV, the causative pathogen of the global SARS outbreak between 2002 to 2004, a neutralizing antibody, namely CR3022, has been identified in SARS recovered patients, and this antibody targets a highly conserved epitope on the RBD of the spike protein.  Recently, CR3022 has been shown to have cross-reactivity against SARS-CoV-2. Some antibodies isolated from COVID-19 patients have been shown to target the CR3022 epitope.

The CR3022 epitope contains 28 amino acid residues, of which 24 are highly conversed between SARS-CoV-2 and SARS-CoV. However, the affinity of CR3022 toward SARS-CoV is 100 times higher than that of SARS-CoV-2. Moreover, a live virus neutralization assay has shown that CR3022 cannot neutralize SARS-CoV-2.

Current study objective  

In the current study, the scientists from the University of Illinois at Urbana-Champaign and The Scripps Research Institute aimed at investing the molecular characteristics justifying the variation in binding affinity and neutralizing efficacy of CR3022 toward SARS-CoV-2 and SARS-CoV.  

Important observations

There are 4 non-conserved residues between the CR3022 epitopes of SARS-CoV-2 and SARS-CoV, which may be responsible for variation in binding affinity of CR3022. To identify the causative variant, the scientists created four mutants of SARS-CoV-2 RBD (A372T, P384A, T430M, and H519N) by substituting the amino acid sequence of CR3022 epitope in SARS-CoV-2 with that of SARS-CoV at four non-conserved residues. Using antibody binding assay, they observed that compared to other mutants, the affinity of CR3022 increased significantly toward the P384A mutant. The level of affinity was similar to that of the CR3022 epitope in SARS-CoV. This indicates that a single amino acid difference at residue 384 is responsible for the variation in CR3022 affinity toward SARS-CoV-2 and SARS-CoV.

The scientists then performed antibody neutralization assay and observed that CR3022 could effectively neutralize the P384A mutant but failed to neutralize wild-type SARS-CoV-2. The level of neutralization was similar to that observed for SARS-CoV. This indicates that because of significantly increased affinity toward the P384A mutant, CR3022 reached the threshold to execute neutralization. With further analysis, the scientists revealed that the neutralization efficiency of CR3022 is more related to its Fab binding affinity.

Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. (A) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor709 sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar (Supplementary Figure 4). The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) [35] is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown.
Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. (A) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor709 sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar. The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) [35] is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown.

X-ray crystallographic analysis revealed structural similarities between the SARS-CoV – CR3022, and SARS-CoV-2 – CR3022 complexes. However, there is a very subtle structural difference in CR3022 elbow angles, responsible for the variation in antigen binding affinity of CR3022. Moreover, the binding of CR3022 to SARS-CoV is energetically more advantageous than binding to SARS-CoV-2.

Further structural analysis using electron microscopy revealed that with three RBDs in the ACE2 receptor accessible state (“up” state), one spike protein of SARS-CoV could bind three CR3022 Fabs simultaneously. In this RBD state, CR3022 was found to make quaternary interaction with the N-terminal domain of a nearby subunit. The scientists also observed that the rotational flexibility of RBD and the acquisition of quaternary interactions are crucial for CR3022 – spike protein interaction.        

Study significance

The current study identifies the key amino acid variant at 384 residue responsible for the variation in binding affinity and neutralizing power of antibodies against SARS-CoV-2 SARS-CoV. Identification of cross-neutralizing epitopes can open up a new path toward the discovery of potential immune-therapeutics or vaccines.  

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Sanchari Sinha Dutta

Written by

Dr. Sanchari Sinha Dutta

Dr. Sanchari Sinha Dutta is a science communicator who believes in spreading the power of science in every corner of the world. She has a Bachelor of Science (B.Sc.) degree and a Master's of Science (M.Sc.) in biology and human physiology. Following her Master's degree, Sanchari went on to study a Ph.D. in human physiology. She has authored more than 10 original research articles, all of which have been published in world renowned international journals.

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