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

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

10.1101/2021.01.14.426475 ◽

2021 ◽

Cited By ~ 7

Author(s):

Matthew McCallum ◽

Anna De Marco ◽

Florian Lempp ◽

M. Alejandra Tortorici ◽

Dora Pinto ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Protective Immunity ◽

Selective Pressure ◽

Memory B Cells ◽

Host Cells ◽

Receptor Binding Domain ◽

Effector Functions ◽

Terminal Domain ◽

Frequent Mutations ◽

A Site

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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ANALYSIS OF IMMUNE ESCAPE VARIANTS FROM ANTIBODY-BASED THERAPEUTICS AGAINST COVID-19.

10.1101/2021.11.11.21266207 ◽

2021 ◽

Author(s):

Daniele Focosi ◽

Fabrizio Maggi ◽

Massimo Franchini ◽

Scott McConnell ◽

Arturo Casadevall

Keyword(s):

Public Health ◽

Monoclonal Antibodies ◽

Immune Evasion ◽

Immune Escape ◽

Selective Pressure ◽

Neutralizing Antibody ◽

Spike Protein ◽

Single Amino Acid ◽

Receptor Binding Domain ◽

Amino Acid Mutations

Accelerated SARS-CoV-2 evolution under selective pressure by massive deployment of neutralizing antibody-based therapeutics is a concern with potentially severe implications for public health. We review here reports of documented immune escape after treatment with monoclonal antibodies and COVID19 convalescent plasma (CCP). While the former is mainly associated with specific single amino acid mutations at residues within the receptor-binding domain (e.g., E484K/Q, Q493R, and S494P), the few cases of immune evasion after CCP were associated with recurrent deletions within the N-terminal domain of Spike protein (e.g, delHV69-70, delLGVY141-144 and delAL243-244). Continuous genomic monitoring of non-responders is needed to better understand immune escape frequencies and fitness of emerging variants.

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Neutralizing antibody-independent immunity to SARS-CoV-2 in hamsters and hACE-2 transgenic mice immunized with a RBD/Nucleocapsid fusion protein

10.1101/2021.09.16.460663 ◽

2021 ◽

Author(s):

Julia Castro ◽

Marcilio Fumagalli ◽

Natalia Hojo-Souza ◽

Patrick Azevedo ◽

Natalia Salazar ◽

...

Keyword(s):

T Cell ◽

Transgenic Mice ◽

Lung Inflammation ◽

Neutralizing Antibodies ◽

Chimeric Protein ◽

Neutralizing Antibody ◽

Receptor Binding Domain ◽

Wild Type ◽

N Protein ◽

Cell Responses

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.

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SARS-CoV-2 immune evasion by variant B.1.427/B.1.429

10.1101/2021.03.31.437925 ◽

2021 ◽

Author(s):

Matthew McCallum ◽

Jessica Bassi ◽

Anna De Marco ◽

Alex Chen ◽

Alexandra C Walls ◽

...

Keyword(s):

Signal Peptide ◽

Immune Evasion ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Clinical Stage ◽

Viral Evolution ◽

Structural Rearrangement ◽

Peptide Cleavage ◽

Terminal Domain ◽

Novel Variant

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.

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Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

10.1101/2021.04.22.441007 ◽

2021 ◽

Author(s):

Marta Alenquer ◽

Filipe Ferreira ◽

Diana Lousa ◽

Mariana Valério ◽

Mónica Medina-Lopes ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Angiotensin Converting Enzyme ◽

Immune Evasion ◽

Neutralizing Antibodies ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Antibody Neutralization ◽

Angiotensin Converting Enzyme 2 ◽

The Core

AbstractUnderstanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike and, predominantly, of its receptor-binding-domain (RBD). Mutations in spike impacting antibody or ACE2 binding are known, but the effect of mutation synergy is less explored. We engineered 22 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent sera. This amino acid emerges as an additional hotspot for immune evasion and a target for therapies, vaccines and diagnostics.One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme 2.

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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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Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

Science ◽

10.1126/science.abi9745 ◽

2021 ◽

pp. eabi9745

Author(s):

Yongfei Cai ◽

Jun Zhang ◽

Tianshu Xiao ◽

Christy L. Lavine ◽

Shaun Rawson ◽

...

Keyword(s):

Immune Evasion ◽

Neutralizing Antibodies ◽

Viral Fitness ◽

Structural Basis ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Antigenic Properties ◽

Structural Details ◽

Fast Spreading

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.

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A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice

10.1101/2020.08.28.272518 ◽

2020 ◽

Cited By ~ 2

Author(s):

Abigail E. Powell ◽

Kaiming Zhang ◽

Mrinmoy Sanyal ◽

Shaogeng Tang ◽

Payton A. Weidenbacher ◽

...

Keyword(s):

Single Dose ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Amino Acid Deletion ◽

Vaccine Candidates ◽

Self Assembling ◽

Antibody Titers

AbstractDevelopment of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

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Structural Basis for Accommodation of Emerging B.1.351 and B.1.1.7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies

10.1101/2021.02.21.432168 ◽

2021 ◽

Cited By ~ 3

Author(s):

Gabriele Cerutti ◽

Micah Rapp ◽

Yicheng Guo ◽

Fabiana Bahna ◽

Jude Bimela ◽

...

Keyword(s):

Monoclonal Antibody ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Structural Basis ◽

Wild Type Virus ◽

Receptor Binding Domain ◽

Type Virus ◽

Wild Type ◽

Distinct Mechanism ◽

The Uk

SummaryEmerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented a response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for both ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies like 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.

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Engineered receptor binding domain immunogens elicit pan-coronavirus neutralizing antibodies

10.1101/2020.12.07.415216 ◽

2020 ◽

Author(s):

Blake M. Hauser ◽

Maya Sangesland ◽

Evan C. Lam ◽

Jared Feldman ◽

Ashraf S. Yousif ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Binding Domain ◽

Surface Glycoprotein ◽

Binding Motif ◽

Receptor Binding Domain ◽

Broadly Neutralizing Antibodies ◽

Major Surface Glycoprotein ◽

Major Surface

AbstractEffective countermeasures are needed against emerging coronaviruses of pandemic potential, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Designing immunogens that elicit broadly neutralizing antibodies to conserved viral epitopes on the major surface glycoprotein, spike, such as the receptor binding domain (RBD) is one potential approach. Here, we report the generation of homotrimeric RBD immunogens from different sarbecoviruses using a stabilized, immune-silent trimerization tag. We find that that a cocktail of homotrimeric sarbecovirus RBDs can elicit a neutralizing response to all components even in context of prior SARS-CoV-2 imprinting. Importantly, the cross-neutralizing antibody responses are focused towards conserved RBD epitopes outside of the ACE-2 receptor-binding motif. This may be an effective strategy for eliciting broadly neutralizing responses leading to a pan-sarbecovirus vaccine.

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