Anti-SARS-CoV-2 NTD-directed antibodies are protective against lethal infection

As the world struggles to contain the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for over 1.6 million deaths and 76 million infections globally, researchers are trying to pin down the determinants of lethality, of infectivity and of protection. Such data would help contain its spread and develop preventive and therapeutic antivirals and vaccines. A new preprint that appeared recently on the bioRxiv* server describes the high degree of protection afforded by antibodies that target the N-terminal domain (NTD) of the virus.

Study: Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma. Image Credit: Kateryna Kon / Shutterstock

Different B cell and plasma cell repertoires

Most antibody research on this virus has focused on single B cells, which are cloned, and their antibodies screened for binding and neutralizing activity in vitro. These show the presence of multiple spike epitopes that are neutralized by a fraction of these monoclonal antibodies (mAbs), mostly those that bind to the viral receptor-binding domain (RBD).

On the other hand, little attention has been paid to the epitopes that are recognized by circulating antibodies in convalescent plasma, and particularly IgG antibodies produced by plasma cells. However, these are a chief part of the neutralizing immune response, and it is important to acknowledge that plasma cells and B cells recognize different epitopes and appear at different time points. This is why anti-SARS-CoV-2 spike or RBD B cells appear at high frequencies along with the presence of powerful neutralizing antibodies, while plasma neutralizing activity remains low.

Study details

Blood samples were obtained from four coronavirus disease 2019 (COVID-19). All were convalescents, having been confirmed to have COVID-19 by reverse-transcriptase polymerase chain reaction (RT PCR). All were 11-19 days from symptom onset.

The researchers found that 84% of the polyclonal IgG antibodies targeted the spike ectodomain (S-ECD) epitopes that lie outside the receptor-binding domain (RBD), including the most abundant plasma IgG lineages in all four samples. The plasma IgG response was oligoclonal in all cases, with 6-22 lineages. Just one lineage accounted for almost a fifth of the whole antibody repertoire.

This agrees with earlier studies, indicating that most mAbs bind to regions of the spike outside the RBD. The IgG Seq tool used here is known to capture 70% of circulating antibodies, and 85% of the most abundant lineages.

NTD antibodies have potent neutralizing activity

In one patient who recovered after just two days of symptoms and who had the lowest plasma neutralizing titers, the researchers found six IgG lineages, of which four made up more than 5% of the total S-ECD antibody set. However, the top two alone made up >70%, and the top four (1 anti-S2, 2 anti-NTD and 1 anti-RBD) made up~94% of the total S-ECD plasma antibodies. The top two also showed abundant diversity within each lineage.

Of these, mAbs CM29-CM32 were the most expanded clones in the top four lineages. CM29, CM30 and CM31 specifically recognized the S2 spike subunit, the NTD and the RBD, respectively. The most powerful neutralization activity was seen with CM30, with CM32 only a little less potent. The latter targets the RBD. The other two, CM29 and CM31, showed insignificant neutralization activity.

In mice, only CM30, the top anti-NTD IgG, showed the ability to protect the mice against infection at low and high doses. CM32 did not protect against infection with a low dose of the virus nor reduce viral loads in the lung. CM29 was also not protective but did reduce viral loads in the lungs, perhaps because it induces antibody-dependent cellular phagocytosis (ADCP) activity. CM31 actually showed a trend towards viral titers in the lung.

Complete protection, with undetectable viral titers in the lung, at high viral doses, was also afforded by a combination of CM29–CM31. Thus, if one potent plasma mAb targeting an epitope outside the RBD is present, it overrides the lack of protection due to the other IgGs in the polyclonal response.

In another patient with more severe and prolonged illness, the top 12 lineages bound to non-RBD epitopes outside the S-ECD, making up more than 80% of the total. The most abundant reacted against the S2 subunit. They also observed three anti-NTD mAbs, which bound the S-ECD at nanomolar affinities, and had potent neutralizing activity against the virus at sub-micromolar titers. These mAbs protected mice against lethal infection and reduced viral titers in the lungs. The most powerful of these is CM25, which has “potency comparable to, or exceeding, clinical-stage antibodies targeting the RBD.”

Thus, the anti-NTD antibody was sufficient for protection against SARS-CoV-2 infection.

Anti-NTD antibodies encoded in IGHV1-24

IGHV refers to the variable region of the variable domain of immunoglobulin heavy chains. The IGHV1-24 is a germline antibody gene that is expressed at low levels in health but at tenfold higher levels with COVID-19, in both B cells and plasmablasts. These antibodies were found to target only S-ECD epitopes outside the RBD.

All three anti-NTD mAbs from the patient with less severe illness, another four from peripheral B cells and another non-neutralizing antibody against the virus, had very similar sequences for immune receptors on the variable regions of the heavy chains. Somatic mutations were very few or absent, however, in this region, including three glutamate residues and one phenylalanine at specific locations. These are unique residues found only in IGHV1-24.

NTD binding occurs at heavy chain

The binding of these IGHV1-24 NTD-reactive antibody interactions with the spike is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area.


This molecular proteomics study of the antibodies targeting SARS-CoV-2 showed an oligoclonal plasma antibody response, mostly directed at the non-RBD epitopes of the S-ECD.

Our report is the first confirmation of the capacity of anti-NTD antibodies to confer protection in vivo and is the first demonstration of convergent antibody recognition of a spike epitope that resides outside the RBD, which collectively point to alternative routes for virus neutralization, the resolution of disease, and provide a rationale for therapeutic interventions based on non-RBD spike epitopes.”

*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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