RBD and NTD antibody cocktail could prevent the emergence of SARS-CoV-2 escape mutants

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has claimed over 3.8 million lives worldwide.

A ray of hope became visible as vaccines received emergency use authorizations (EUAs). Simultaneously, however, new variants of the virus have emerged, some forming variants of concern (VOCs) that are either more infective or show increased resistance to antibody-mediated neutralization.

A new bioRxiv* preprint demonstrates how a combination of antibodies to two different regions on the viral spike could enhance neutralization efficiency as well as prevent the emergence of resistant variants.


The virus mediates entry into host cells via its transmembrane spike antigen, a glycoprotein that is found as a trimer. Each monomer is composed of S1 and S2 subunits and must be activated by host protease enzymes.

The S1 subunit has an N-terminal domain and (NTD) and a C-terminal domain (CTD). The latter contains the receptor-binding domain (RBD) that binds the host cell receptor, the human angiotensin-converting enzyme 2 (ACE2).

The spike protein is the immunodominant antigen and is targeted by the majority of neutralizing antibodies (NAbs) as part of the immune response induced by natural infection or vaccination. Much attention has been paid to antibodies directed against the RBD since these neutralize infection by disrupting virus-receptor interaction.

Monoclonal NAbs targeting the RBD have also received EUAs, but some have since lost their approval because of their lack of efficacy against VOCs in circulation. This spurred research into combinations of NAbs to treat the infection effectively while minimizing the chances of mutational escape.

Neutralizing antibodies to the RBD and the NTD

In the current study, the researchers identified RBD- and NTD- specific NAbs in the activated memory B cells sets of four convalescent COVID-19 donors with high antibody titers against the viral spike. The most potent NAbs against both RBD and NTD were selected and their epitopes defined.

The study showed that there was an early strong memory B cell response to the virus in all donors. There were >40 monoclonal antibodies (MAbs) that had over 50% neutralization efficiency against the authentic virus and >3 against SARS-CoV-2 spike-expressing pseudovirus.

Most NAbs targeted the RBD, inhibiting the ACE2 receptor either competitively or non-competitively, but there were also several NTD-directed MAbs. The top anti-RBD antibodies had picomolar concentrations at their half-maximal inhibitory concentration (IC50) against both pseudovirus and authentic SARS-CoV-2.

Escape mutations

Anti-RBD antibody epitope mapping from the pseudovirus spike mutations that conferred antibody escape showed that a change at F490 to L, I or C residues boosted antibody resistance, as indicated by >2,700-fold higher IC50 values for the top anti-RBD antibody ADI-56443. RBD binding disappeared with mutations at C480 (to S/R), E484 (to K/G/D), and C488 (toY/S) as well.

Reduced, though not absent, binding was found with the S494F mutation in the RBD. This antibody shows some overlap with the RBD-binding interface.

Interestingly, many VOCs and variants such as P.1, P.2, 232 B.1.525 and B.1.351 carry E484K, indicating resistance to currently used MAbs. E484, along with F490, and S494, are key residues present in the epitope bound by mAb LY-CoV555 (Bamlanivimab), used to treat COVID-19, which is thereby rendered ineffective in the presence of mutations at these locations.

With the anti-NTD antibodies, complete neutralization was achieved only with the authentic virus, at picomolar concentrations, some of the pseudoviral particles escaping neutralization.

Mutational escape to the NTD mAb ADI-56479 occurred with Y145D, K150E and W152R mutations, each of which increased the IC50 a thousand-fold.

These mutations also caused loss of binding to the spike protein on pseudovirus particles and are resistant to neutralization of pseudoviruses by sera from convalescent COVID-19 patients. Y145D caused neutralization efficiency to drop by 3.5-fold, while K150E and W152R increased the IC50 by 16-fold, compared to the parental virus strain.

These residues are found away from the RBD binding interface, on an antigenic supersite, and are bound by other powerful anti-NTD NAbs as well.

A W152 mutation is also found in the VOC B.1.429, which is also more resistant to neutralization by antibodies in vaccinated sera or convalescent sera. VOC B.1.617.2 also has mutations (deletions) at positions 156 and 157.

This indicates that NTD binding is important in neutralizing SARS-CoV-2 in COVID-19 and that escape mutations are being accumulated in response. Other mutations at sites such as N148S, K150R/E and S151P also reduced the sensitivity of the virus to convalescent serum.

Benefits of combining antibodies

The combination of these antibodies was found to enhance the efficiency with which they neutralized the virus. However, the degree of synergism could not be calculated owing to their very different IC50 values. When both were combined at their IC50 doses, the cocktail limited the emergence of escape mutants to 72 infectious units per mL.

Conversely, the use of a single MAb led to the rapid emergence of 105 to 106 infectious units per mL, compared to 107 for controls, at 48 hours after challenge with the virus.

What are the implications?

The RBD antibody ADI-56443 and the NTD antibody ADI-56479 make up a combination that bind to two different locations on the viral spike, with a moderate improvement in efficiency as well as limiting the emergence of NAb-resistant spike mutations.

The scientists found that most of the top NTD NAbs were generated early in the infection, indicating that their ability to effectively counter the virus was independent of high-affinity binding. Their mechanism of inhibition may be linked to inhibition of viral replication post-attachment or the activation of Fc-mediated effector functions.

With evidence that some NTD-binding MAbs play a major part in neutralizing SARS-CoV-2, and that escape mutations are emerging within VOCs, “a combination antibody therapy with RBD and NTD nAbs could provide an important solution for treating COVID-19 and impede the generation of novel variants especially in populations that cannot be vaccinated.”

*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. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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