N-terminal domain antigenic mapping reveals a site of vulnerability for SARS-CoV-2

SARS-CoV-2 entry into host cells is orchestrated by the spike (S) glycoprotein that contains an immunodominant receptor-binding domain (RBD) targeted by the largest fraction of neutralizing antibodies (Abs) in COVID-19 patient plasma. Little is known about neutralizing Abs binding to epitopes outside the RBD and their contribution to protection. Here, we describe 41 human monoclonal Abs (mAbs) derived from memory B cells, which recognize the SARS-CoV-2 S N-terminal domain (NTD) and show that a subset of them neutralize SARS-CoV-2 ultrapotently. We define an antigenic map of the SARS-CoV-2 NTD and identify a supersite recognized by all known NTD-specific neutralizing mAbs. These mAbs inhibit cell-to-cell fusion, activate effector functions, and protect Syrian hamsters from SARS-CoV-2 challenge. SARS-CoV-2 variants, including the 501Y.V2 and B.1.1.7 lineages, harbor frequent mutations localized in the NTD supersite suggesting ongoing selective pressure and the importance of NTD-specific neutralizing mAbs to protective immunity.

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Neutralizing antibody 5-7 defines a distinct site of vulnerability in SARS-CoV-2 spike N-terminal domain

10.1101/2021.06.29.450397 ◽

2021 ◽

Author(s):

Gabriele Cerutti ◽

Yicheng Guo ◽

Pengfei Wang ◽

Manoj S Nair ◽

Yaoxing Huang ◽

...

Keyword(s):

Immune Evasion ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Receptor Binding Domain ◽

Hydrophobic Pocket ◽

Terminal Domain ◽

Distinct Site ◽

Binding Antibody ◽

A Site ◽

Binding Loop

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD- directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the 3.7 Å resolution cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD β-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition.

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Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

Science Translational Medicine ◽

10.1126/scitranslmed.abd6990 ◽

2021 ◽

pp. eabd6990

Author(s):

Sang Il Kim ◽

Jinsung Noh ◽

Sujeong Kim ◽

Younggeun Choi ◽

Duck Kyun Yoo ◽

...

Keyword(s):

B Cells ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Spike Protein ◽

Receptor Binding Domain ◽

Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

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Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity

10.1101/2021.04.26.441518 ◽

2021 ◽

Author(s):

William E. Matchett ◽

Vineet Joag ◽

J. Michael Stolley ◽

Frances K. Shepherd ◽

Clare F. Quarnstrom ◽

...

Keyword(s):

Weight Loss ◽

Viral Load ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Protein A ◽

Receptor Binding Domain ◽

Immune Effector ◽

N Protein ◽

Viral Antigens ◽

Successful Vaccine

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

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Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1609316113 ◽

2016 ◽

Vol 113 (42) ◽

pp. 11931-11936 ◽

Cited By ~ 88

Author(s):

Wenqian He ◽

Gene S. Tan ◽

Caitlin E. Mullarkey ◽

Amanda J. Lee ◽

Mannie Man Wai Lam ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A Virus ◽

Neutralizing Antibodies ◽

Influenza A ◽

Critical Role ◽

Host Cells ◽

Viral Glycoprotein ◽

Effector Functions ◽

Dependent Cell

The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

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Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

10.1101/2021.02.03.429355 ◽

2021 ◽

Author(s):

Carl Graham ◽

Jeffrey Seow ◽

Isabella Huettner ◽

Hataf Khan ◽

Neophytos Kouphou ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Viral Entry ◽

Neutralizing Antibodies ◽

Infectious Virus ◽

Antigenic Drift ◽

Host Cells ◽

Receptor Binding Domain ◽

Neutralization Activity ◽

Neutralizing Activity ◽

Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

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The self-assembled nanoparticle-based trimeric RBD mRNA vaccine elicits robust and durable protective immunity against SARS-CoV-2 in mice

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00750-w ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Wenqiang Sun ◽

Lihong He ◽

He Zhang ◽

Xiaodong Tian ◽

Zhihua Bai ◽

...

Keyword(s):

Receptor Binding ◽

Humoral Immunity ◽

Broad Spectrum ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Cellular Response ◽

Low Dose ◽

The Self ◽

Receptor Binding Domain ◽

Self Assembled

AbstractAs COVID-19 continues to spread rapidly worldwide and variants continue to emerge, the development and deployment of safe and effective vaccines are urgently needed. Here, we developed an mRNA vaccine based on the trimeric receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein fused to ferritin-formed nanoparticles (TF-RBD). Compared to the trimeric form of the RBD mRNA vaccine (T-RBD), TF-RBD delivered intramuscularly elicited robust and durable humoral immunity as well as a Th1-biased cellular response. After further challenge with live SARS-CoV-2, immunization with a two-shot low-dose regimen of TF-RBD provided adequate protection in hACE2-transduced mice. In addition, the mRNA template of TF-RBD was easily and quickly engineered into a variant vaccine to address SARS-CoV-2 mutations. The TF-RBD multivalent vaccine produced broad-spectrum neutralizing antibodies against Alpha (B.1.1.7) and Beta (B.1.351) variants. This mRNA vaccine based on the encoded self-assembled nanoparticle-based trimer RBD provides a reference for the design of mRNA vaccines targeting SARS-CoV-2.

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Xeno-Nucleic Acid (XNA) 2’-Fluoro-Arabino Nucleic Acid (FANA) Aptamers to the Receptor-Binding Domain of SARS-CoV-2 S Protein Block ACE2 Binding

Viruses ◽

10.3390/v13101983 ◽

2021 ◽

Vol 13 (10) ◽

pp. 1983

Author(s):

Irani Alves Ferreira-Bravo ◽

Jeffrey J. DeStefano

Keyword(s):

Nucleic Acid ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Dissociation Constants ◽

High Specificity ◽

Binding Domain ◽

Host Cells ◽

Receptor Binding Domain ◽

S Protein ◽

Equilibrium Dissociation Constants

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2ʹ-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10–20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.

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Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2

10.1101/2021.02.17.431722 ◽

2021 ◽

Author(s):

Huibin Lv ◽

Owen Tak-Yin Tsang ◽

Ray T. Y. So ◽

Yiquan Wang ◽

Meng Yuan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Global Economy ◽

Vaccine Development ◽

Serological Response ◽

Receptor Binding Domain ◽

Plasma Samples ◽

Mice Model ◽

Terminal Domain ◽

Major Antigen ◽

Serology Testing

SUMMARYThe increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the serological response to SARS-CoV-2 RBD, the SARS-CoV-2 NTD response is less cross-reactive with SARS-CoV. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers an alternative strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

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Development of spike receptor-binding domain nanoparticle as a vaccine candidate against SARS-CoV-2 infection in ferrets

10.1101/2021.01.28.428743 ◽

2021 ◽

Author(s):

Young-Il Kim ◽

Dokyun Kim ◽

Kwang-Min Yu ◽

Hogyu David Seo ◽

Shin-Ae Lee ◽

...

Keyword(s):

Receptor Binding ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Clinical Symptoms ◽

Binding Domain ◽

Host Cells ◽

Antigen Delivery ◽

Receptor Binding Domain ◽

Protein Vaccine

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of COVID-19 pandemic, enters host cells via the interaction of its Receptor-Binding Domain (RBD) of Spike protein with host Angiotensin-Converting Enzyme 2 (ACE2). Therefore, RBD is a promising vaccine target to induce protective immunity against SARS-CoV-2 infection. In this study, we report the development of RBD protein-based vaccine candidate against SARS-CoV-2 using self-assembling H. pylori-bullfrog ferritin nanoparticles as an antigen delivery. RBD-ferritin protein purified from mammalian cells efficiently assembled into 24-mer nanoparticles. 16-20 months-old ferrets were vaccinated with RBD-ferritin nanoparticles (RBD-nanoparticles) by intramuscular or intranasal inoculation. All vaccinated ferrets with RBD-nanoparticles produced potent neutralizing antibodies against SARS-CoV-2. Strikingly, vaccinated ferrets demonstrated efficient protection from SARS-CoV-2 challenge, showing no fever, body weight loss and clinical symptoms. Furthermore, vaccinated ferrets showed rapid clearance of infectious viruses in nasal washes and lungs as well as viral RNA in respiratory organs. This study demonstrates the Spike RBD-nanoparticle as an effective protein vaccine candidate against SARS-CoV-2.

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Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

10.1101/2021.02.02.428995 ◽

2021 ◽

Author(s):

Tal Noy-Porat ◽

Adva Mechaly ◽

Yinon Levy ◽

Efi Makdasi ◽

Ron Alcalay ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Phage Display Library ◽

Host Cells ◽

Terminal Domain ◽

Isolation And Characterization ◽

Recent Emergence ◽

Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

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