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A novel neutralizing antibody targeting the N-terminal domain of SARS-CoV-2 was discovered

 


The main purpose of scientific research on treatments to combat the pandemic of ongoing coronavirus disease 2019 (COVID-19) is the invasion of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV). It is to identify drugs and antibodies that can act on the disease. -2) In the host cell.

A new study by US-based researchers shows the presence of protective antibodies that target the N-terminal domain (NTD) of spikes rather than the spike receptor binding domain (RBD).

Study: Neutralizing and protecting human monoclonal antibody that recognizes the N-terminal domain of SARS-CoV-2 peplomer. Image Credit: ktsdesign / Shutterstock

Vanderbilt Vaccine Center, University of Washington School of Medicine, St. Louis, Integral Molecular Team – Preprint bioRxiv* server.

Spike protein

Viral invasion is mediated by viral peplomers that bind to the host cell receptor angiotensin converting enzyme 2 (ACE2) via RBD.Spikes are trimers Glycoprotein Each protomer has two subunits, S1 and S2. S1 contains RBD and is responsible for binding ACE2.

The S2 subunit mediates the membrane fusion of the virus and the cell and the invasion of the virus into the cell.most Neutralizing antibody Therefore, we are targeting spike RBD.

The viral RNA genome and protein transcriptome have changed thousands of times, with an average of four mutations per amino acid in peplomer proteins. This raised concerns about whether viral variants would allow escape from neutralizing antibodies. Most neutralizing antibodies bind to RBD. Some scientists have reported monoclonal antibodies (mAbs) that also bind to the outer region of the RBD.

Neutralizing effect

Current studies identify the structure and function of three anti-NTD antibodies selected from a library of approximately 390 mAbs obtained from individuals who have recovered from natural SARS-CoV-2 infection. Two of the three had strong neutralizing abilities. These were named COV2-2676 and COV2-2489.

They used two neutralization assays, a focus-reducing neutralization test (FRNT) using live virus and a second real-time cell analysis (RTCA) using vesicular stomatitis virus capable of spiking expression recombination replication. Assay (VSV) pseudovirus. They found that both mAbs were able to neutralize the FRNT wild-type virus in a dose-dependent manner, with half of the maximum inhibition (IC50) values ​​being 501 or 199 ng / mL, respectively. In the second example, both neutralized the pseudovirus and the IC50 was 8 or 56 ng / mL, respectively.

Common binding area

Scientists have identified epitopes that bind to these potent neutralizing antibodies. Both bound to distinct antigenic sites as compared to epitopes bound by anti-RBD human mAbs. Both of these antibodies were unable to prevent soluble ACE2-soluble RBD binding. In fact, both antibodies bound to spike NTDs in a “closed” conformation of the spike trimer. They compete fiercely with each other for a common binding epitope. Both are independent chronotypes and are encoded by the heavy chain variable gene segments IGHV-4-39 and IGHV1-69, respectively. However, the HCDR3 area is quite different.

Recognition of SARS-CoV-2 spike NTD

The heavy loops of both of these antibodies interact with spiked NTDs in the N3 and N5 loops, respectively, which is a potential therapeutic target, and individuals with acquired immunity to the virus produce antibodies that target this region. Is shown.

Using an alanine scan, researchers also determined that a single alanine change at several positions involved in the binding of these mAbs does not impair the binding of another anti-NTD antibody COV2-2305. Did. .. Using the RTCA assay, they found escape mutations that resist neutralization with these antibodies at levels of COV2-2676 and COV2-2489 at 10 or 100 μg / mL, respectively. The escape mutation was in the same region identified by the alanine scan. Thus, both methods show a common antigenic site that binds to both antibodies, having different epitopes within this region.

Spike binding to infected cells

They found that these mAbs bind enthusiastically Spike protein It exhibits higher efficiency and density of binding on the surface of infected cells compared to antibodies targeting RBD. They repeated FRNT on several cell lines cultured with the virus. However, they found that this binding was different for different cell types, probably because the viral invaders and receptors differ between different types of cells.

They also compared the results of incubating mAbs with the virus before and after absorption on the cell surface. They found that the use of these antibodies resulted in strong neutralization both before and after attachment to the virus. These findings suggest that the virus can be used to prevent infection even after it has attached to cells.

Anti-RBD antibodies inhibited viral attachment, but no neutralization was observed with these mAbs in cells overexpressing human ACE2 and TMPRSS2.

Requires full length or F (ab’) 2 form

Both of these mAbs were able to neutralize the virus only in the form of full-length IgG or divalent Fab, but neither monovalent Fab fragment was able to neutralize the virus. In contrast, the strongly neutralizing anti-RBD antibody Fab inhibited infection as efficiently as full-length antibody.

Overlaid Fab-Spike negative stain EM.  A) COV 2-2676 with mAb at the top is side view 4A8 (left), mAb 4-8 (right), and the bottom is the same top view.  B) COV 2-2489 with mAb at the top is side view 4A8 (left), mAb 4-8 (right), and the bottom is the same top view.

Overlaid Fab-Spike negative stain EM. A) COV 2-2676 with mAb at the top is side view 4A8 (left), mAb 4-8 (right), and the bottom is the same top view. B) COV 2-2489 with mAb at the top is side view 4A8 (left), mAb 4-8 (right), and the bottom is the same top view.

The researchers said,Larger IgG or F (ab’) 2 can sterically interfere with the functional interaction of S proteins during entry.Alternatively, the monovalent Fab molecule may have too low an affinity for NTD to inhibit binding... “

Mechanism via Fc

These antibodies required intact Fc-mediated function for their activity In vivo.. When these interactions were abolished, mice showed greater weight loss, higher viral load, and higher lung inflammation.

Mouse protection effect

Researchers have found that these mAbs protect mice that express the human ACE2 receptor when administered before and after a viral attack. Treated mice showed no weight loss, reduced viral load, and reduced cytokine levels, similar to the effects of anti-RBD antibodies.

What is the impact?

The region of NTD is recognized by neutralizing and protective antibodies against SARS-CoV-2 and can function as part of the antibody cocktail to minimize the choice of resistance to escape or natural variants of RBD. I will. “

Researchers recommend using a combination of spike RBD and NTD-targeting antibodies to increase defense levels and reduce the risk of escape mutations, but while provided by anti-NTD antibodies. The potency of sum is the most powerful neutralizing anti-RBD antibody. Vaccines may also provide the most durable immunity when developed against multiple antigens, not just spike RBD.

*Important Notices

bioRxiv Publish preliminary scientific reports that should not be considered definitive as they are not peer-reviewed, guide clinical practice / health-related behaviors, and should not be treated as established information.

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