Avidity of binding responsible for cross-neutralization of SARS-CoV-1 and SARS-CoV-2

By Dr. Liji Thomas, MDAug 5 2020

The current pandemic of COVID-19 will need effective antiviral strategies as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread is unlikely to be countered by non-pharmacological interventions completely.

At present, many researchers are looking into developing vaccines that generate a neutralizing antibody response, mostly targeting the receptor-binding domain (RBD) on the spike protein of the SARS-CoV-2 virus. This is the entry protein for the virus into the human host cell, attaching the virus to the human angiotensin-converting enzyme (ACE)2 receptor. This attachment triggers further viral-host cell membrane fusion, endocytosis, and then cleavage of the spike protein to allow the virus to escape the endosome and enter the cytoplasm of the host cell, to initiate replication and infection.  

A Highly Conserved Epitope

Both SARS-CoV-1 and SARS-CoV-2 are members of the same phylogenetic tree, with 73% identical amino acid sequences in the RBD region but 90% in the S2 fusion domain. However, prior research has shown that the SARS-CoV-2 RBD carries a highly conserved epitope, found to exist from the study of CR3022, an antibody that neutralizes SARS-CoV-1, and was first isolated 15 years ago.  

Cross-Reactive Antibodies

Cross-reactive antibodies to both SARS-CoV and SARS-CoV-2 are rare, only three having been structurally characterized so far. Such antibodies need to be well understood to design better vaccines and drugs that will protect not only against the current virus but similar emerging viruses in the future.  

One of these cross-reactive antibodies is COVA1-16, recovered from a convalescent COVID-19 patient. It neutralizes both viruses at micromolar concentrations, but the neutralizing concentration needed for SARS-CoV is 20 times higher than for the current virus. COVA1-16 has several heavy and light chains and a long complementarity 66 determining region (CDR) H3 of 20 amino acids.  

Comparison of COVA1-16 binding mode with CR3022 and ACE2. (A) Crystal structure of COVA1-16/RBD complex with RBD in grey and COVA1-16 Fab in cyan (heavy chain) and greyish blue (light chain). (B) ACE2-binding site (PDB 6M0J, left) [10], COVA1- 16 epitope (this study, middle), and CR3022 epitope (PDB 6W41, right) [13] are highlighted in yellow. (C) RBD residues in the COVA1-16 epitope are shown. Epitope residues contacting the heavy chain are in orange and light chain in yellow. Representative epitope residues are labeled. Residues that are also part of CR3022 epitope are indicated with asterisks. (D) The ACE2/RBD complex structure is aligned in the same orientation as the COVA1-16/RBD complex. COVA1-16 (cyan) would clash with ACE2 (green) if they were to approach their respective RBD binding sites at the same time (indicated by red circle).

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Crystal Structure of COVA1-16

In a new study published on the preprint server bioRxiv* researchers characterized the crystal structure of the antibody-SARS-CoV-2 RBD complex in an attempt to find the epitope and the mechanism that enables cross-neutralization. This study showed extensive overlap between the epitopes binding this antibody and CR3022, at 17/25 and 17/28 amino acid residues. However, the former is more extensive, with a region towards the border of the binding site for ACE2.  

Steric Hindrance vs. Overlapping Epitopes

This means that COVA1-16 can bind RBD competitively with CR3022 and also with ACE2, surprisingly, despite its epitope being outside the ACE2 binding site. It is also somewhat similar to another cross-neutralizing antibody ADI-56046 which also binds to a region overlapping both the CR3022 epitope and the ACE2 binding site.

The researchers conclude, “COVA1-16 inhibits ACE2 81 binding due to steric hindrance with its light chain rather than by direct interaction with the 82 receptor binding site.”

As is well-known, the RBD has both up and down conformations, only the former being susceptible to ACE2 binding. However, earlier identified cross-neutralizing antibodies could bind to both conformations. The current antibody COVA-1-16 is different in that it can bind only in the up conformation, being wholly hidden like CR3022 in the down conformation. However, the binding epitope for COVA1-16 is partly hidden even when the RBD is in the up conformation unless the S protein is bound to a ligand.  

Binds at Various Orientations

This prompted the researchers to delve deeper, finding that the antibody is very flexible, capable of binding to the RBD at several orientations, such as perpendicularly to the top of the spike trimeric protein or in a more tilted fashion to the side of the RBD. It is also quite possible that the antibody attaches two RBDs simultaneously since it is a bivalent molecule. This is supported by the fact the binding strength is 40 times greater for the IgG form of COVA1-16 compared to the Fab form, but went down when the amount of RBD was reduced, indicating bivalent binding might be occurring.  

The Importance of Bivalence

Bivalence is also important for the neutralizing capacity of this antibody since the measured IC50 (the concentration at which inhibition is half of the maximal) for COVA1-16 is similar to that measured in earlier studies using SARS-CoV-2 pseudovirus. However, the Fab form fails to show neutralization at 100 times higher concentrations. Interestingly, this antibody fails to show similar neutralizing potency for the actual virus, perhaps because of the lower expression of S on the virus compared to the pseudovirus, or because of differences in the number and conformation of S proteins on the virus. This aspect needs to be further studied.  

COVA1-16 Binding to RBD

The heavy chain is responsible for most of the antibody’s binding to the viral RBD, making up over 80% of the total buried area.  In fact the CDR H3 makes up 70% of this area and takes part in most of the antibody-epitope interactions. The researchers studied the structure of this region, finding 5 hydrogen bonds, four in the main chain, and one in the side chain. Four of these interact with the main chain of the RBD and two with the side chain. There are also four other hydrogen bonds formed between the CDR H3 and the RBD side chains. Structural visualization showed the unliganded structure of the antibody to have an unresolved distal region indicating that it is a flexible region.  

Interaction between SARS-CoV-2 RBD and structurally characterized antibodies. The binding of known SARS-CoV-2 RBD-targeting antibodies to the RBD is compared. The ACE2-binding site overlaps with epitopes of B38 (PDB 7BZ5), C105 (6XCM), CB6 (7C01), CC12.1 (6XC3), CC12.3 (6XC4), BD23 (7BYR) [7], and P2B-2F6 (7BWJ), but not the epitopes of COVA1-16 (this study), CR3022 (PDB 6W41), COVA2-04, COVA2-39], and S309 (PDB 6WPS). Of note, while CR3022 only neutralizes SARS-CoV but not SARS-CoV-2 in in vitro assays, a recent study isolated an antibody (EY6A) that binds to a similar epitope as CR3022 and cross-neutralizes SARS-CoV-2 and SARS-CoV.

The Importance of This Study

Other viruses have also shown antibodies that use the CDR H3 to bind to pathogens, such as the HIV antibodies PG9 and PG16, and the influenza antibody C05. Thus, COVA1-16 could help design further therapeutics.  

The highly conserved nature of the epitope to which COVA1-16 binds may be explained by the mixture of electrostatic forces, both attractive and repulsive, which make the protein metastable. This is essential to allow the RBD to flip up and down in the prefusion state and the overall regulation of the dynamics of the RBD. In other words, the control of the biological function of the prefusion S protein for both receptor binding and fusion is dependent on this epitope structure. Moreover, it is complementary in shape, fitting the other RBDs as well as the S2 domain of the trimeric spike protein. All these may explain the hidden nature as well as the high degree of conservation of this epitope.

Finally, the inaccessibility of the epitope may mean that the S-based immunogen is more potent at eliciting a higher number of antibodies like COVA1-16.

The researchers sum up, “These findings, along with structural and functional rationale for the epitope conservation, provide a blueprint for development of more universal SARS-like coronavirus vaccines and therapies.”

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources