COVID-19: Discovery of New Spike Protein Neutralizing Antibody Reveals Promising Drug Target
By Sahana Shankar, Ph.D. Candidate
As opposed to antibodies that bind to the receptor-binding domain, scientists discover a new antibody that binds to the N-Terminal Domain of the SARS-CoV-2’s spike protein, potentially uncovering a novel drug target.
While the global COVID-19 vaccine efforts are advancing at full throttle, data from recovering patients have facilitated the development of ancillary treatments such as convalescent plasma, neutralizing antibodies, and repurposed drugs such as remdesivir and dexamethasone. Many clinicians have expressed optimism about the use of neutralizing antibodies as an interim measure while we await the vaccine.
It is now established that the COVID-19 causing SARS-CoV-2 uses its spike (S) protein to gain cellular entry. The cell surface protein is composed of 2 subunits, S1, and S2, both of which constitute the extracellular domain (ECD). While the N-terminal domain (NTD) and the receptor-binding domain (RBD) of S1 enable receptor binding, S2 mediates membrane fusion to deliver viral RNA into the host. At present, the conventionally accepted pathway of SARS-CoV-2 infection is via the binding of RBD to the ACE2 receptor on host cells. However, new reports have also suggested a secondary pathway via the NRP1 receptor.
The immune system of recovering patients identifies different parts of the viral proteins as antigens and develops antibodies against them. The neutralizing ability can vary between individuals based upon the specific antigens the antibodies target. Since they exert an effective and specific antiviral response, they can be isolated and characterized to develop therapeutics. Quite a few studies have focused on neutralizing antibodies against the RBD. However, results suggest that a cocktail of antibodies targeting different viral proteins would be more robust and effective for viral clearance.
In a new collaborative study published in Science, scientists from Beijing report the high neutralization potency of a monoclonal antibody against the NTD of the S protein. Plasma and peripheral blood mononuclear cells (PBMCs) of ten recovered Chinese patients were evaluated for their binding affinity to the nucleocapsid (N) protein and the ECD, S1, S2, and RBD domains of the S protein. Plasma antibodies from nine patients were able to bind all five protein fragments, and the remaining one was able to bind to ECD and S2 domains. These Abs were able to neutralize HIV-vectored SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 in Vero6 cells, demonstrating their ability to elicit an immune response against SARS-CoV-2.
Of the 399 monoclonal Abs (mAbs) isolated by sorting IgG+ memory B cells and plasma B cells, the team screened for S protein-specific antibodies and classified them into four groups based on EC50 values by ELISA.
With a competition-binding assay, they screened for overlapping antigenic sites shared between mAbs from different groups and found that there was a significant diversity in the epitopes recognized, indicating that the SARS-CoV-2 infection induces a wide range of antibody responses.
To further characterize the activity, they performed neutralization studies on 35 mAbs against S-ECD and found three candidates with high to medium neutralizing capacity. Interestingly, antibodies against RBD did not have high inhibitory activity. With real-time qPCR, cytopathic effect assay, and luciferase reporter assays, the investigators confirmed their antiviral activity. Interestingly, two of them were able to protect ACE2-293T cells from SARS-CoV-2 pseudovirus but were not effective against the authentic SARS-CoV-2 virus. Thus, 4A8 mAb emerged as a potent antibody against SARS-CoV-2 and provided a potential drug candidate.
To further understand the binding mechanism, the protein complex was subjected to structural studies. In concordance with previous reports, the cryo-EM structure at 3.1Å showed that the trimeric S protein has two asymmetric conformations, two of the three RBDs in ‘down’ conformation and one in ‘up’ conformation. The trimeric S protein was bound to 3 4A8 Fab chains by its NTD. However, irrespective of the up/down conformations, the binding interface between the NTD and 4A8 was identical. Besides, the 4A8 binding stabilized the NTD and improved the structural model by identifying five new loops, adding more information. Thanks to the high resolution, specific residues promoting the interactions between the antibody chains, and helical regions of the NTD were identified.
The distinguishing feature of the study is that the majority of mAbs did not recognize the RBD of the spike protein, which is thought to be the most promising drug target. Remarkably, these mAbs did not prevent S protein binding to ACE2, indicating there are other mechanisms to block viral entry. In conclusion, the study proposes that the NTD of the S protein may be a vulnerable epitope and may be a suitable candidate for a structure-based vaccine. The 4A8 mAb could lead to an ‘antibody cocktail,’ which could further enhance the antiviral activity against SARS-CoV-2.
Editor: Rajaneesh K. Gopinath, Ph.D.
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