Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies

Understanding 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. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We 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 and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

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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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In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

International Journal of Molecular Sciences ◽

10.3390/ijms22041695 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1695

Author(s):

Bruno O. Villoutreix ◽

Vincent Calvez ◽

Anne-Geneviève Marcelin ◽

Abdel-Majid Khatib

Keyword(s):

Amino Acid ◽

South African ◽

In Silico ◽

Neutralizing Antibodies ◽

Viral Escape ◽

Spike Protein ◽

Amino Acid Substitutions ◽

The South ◽

African Strain ◽

The Uk

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.

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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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Construction and immunogenic studies of a mFc fusion receptor binding domain (RBD) of spike protein as a subunit vaccine against SARS-CoV-2 infection

Chemical Communications ◽

10.1039/d0cc03263h ◽

2020 ◽

Vol 56 (61) ◽

pp. 8683-8686 ◽

Cited By ~ 5

Author(s):

Xiaoxiao Qi ◽

Bixia Ke ◽

Qian Feng ◽

Deying Yang ◽

Qinghai Lian ◽

...

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Subunit Vaccine ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Humoral And Cellular Immunity ◽

A Subunit

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice.

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Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Nature Immunology ◽

10.1038/s41590-021-01068-z ◽

2021 ◽

Author(s):

Vincent Dussupt ◽

Rajeshwer S. Sankhala ◽

Letzibeth Mendez-Rivera ◽

Samantha M. Townsley ◽

Fabian Schmidt ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Low Dose ◽

Viral Escape ◽

Spike Protein ◽

Receptor Binding Domain ◽

Viral Inactivation ◽

Angiotensin Converting Enzyme 2 ◽

Therapeutic Monoclonal Antibodies ◽

Potent Protection

AbstractPrevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

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Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike based SARS-CoV-2 vaccine in the mouse model

10.1101/2020.09.16.300970 ◽

2020 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Raveen Rathnasinghe ◽

Michael Schotsaert ◽

Lynda Coughlan ◽

...

Keyword(s):

Mouse Model ◽

Cleavage Site ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Middle Eastern ◽

Host Cells ◽

Spike Protein ◽

Optimized Design ◽

Angiotensin Converting Enzyme 2 ◽

Viral Target

AbstractThe spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively instable, a feature that might be enhanced by the presence of a polybasic cleavage site in the SARS-CoV-2 spike. Exchange of K986 and V987 to prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle Eastern respiratory syndrome coronavirus spikes. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin converting enzyme 2 via adenovirus transduction. Variants tested include spike protein with a deleted polybasic cleavage site, the proline mutations, a combination thereof, as well as the wild type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the PP mutations completely protected from challenge in this mouse model.ImportanceA vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validates the choice of antigens that contain the PP mutation and suggests that deletion of the polybasic cleavage site could lead to a further optimized design.

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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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Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients

10.21203/rs.3.rs-38173/v2 ◽

2020 ◽

Author(s):

Lisu Huang ◽

Bingqing Shen ◽

Yu Guo ◽

Shu Shen ◽

Heyu Huang ◽

...

Keyword(s):

B Cell ◽

Neutralizing Antibodies ◽

Therapeutic Option ◽

Antiviral Drug ◽

Cell Receptor ◽

High Serum ◽

Spike Protein ◽

Live Virus ◽

Angiotensin Converting Enzyme 2 ◽

Humoral Responses

Abstract The pandemic Coronavirus Disease 2019 (COVID-19) causes noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibodies for treatment purposes is a viable alternative. In this study, we aimed to profile the humoral responses and identify neutralizing antibodies against SARS-CoV-2 using high-throughput single-cell sequencing that tailored to B cell receptor sequencing. From two convalescent patients with high serum titer against SARS-COV-2, we identified seven antibodies specifically binding to SARS-CoV-2. Among these, the most potent antibody, P4A1 was demonstrated to block the binding of spike protein to its receptor angiotensin-converting enzyme 2 (ACE2), and prevent the viral infection in neutralization assays with pseudovirus as well as live virus at nM to sub-nM range. Moreover, antibody P4A1 can also bind strongly to spike protein with N354D/D364Y, R408I, W436R, V367F or D614G mutations respectively, suggesting that the antibody alone or in combination with other antibodies that recognize different variations of SARS-CoV-2, may provide a broad spectrum therapeutic option for COVID-19 patients. Authors Lisu Huang, Bingqing Shen, Yu Guo, and Shu Shen contributed equally to this work.

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Protein Footprinting, Conformational Dynamics and Interface-Adjacent Neutralization ‘Hotspots’ in the SARS-CoV-2 Spike Protein Receptor Binding Domain / Human ACE2 Interaction

10.26434/chemrxiv.13385387 ◽

2020 ◽

Author(s):

Dominic Narang ◽

Matthew Balmer ◽

D. Andrew James ◽

Derek Wilson

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Conformational Dynamics ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Additional Information ◽

Protein Receptor ◽

Hdx Ms

This study provides an HDX-MS based analysis of the interaction between the SARS-CoV-2 spike protein and the human Angiotensin Converting Enzyme 2. <div><br></div><div>- The data agree exactly with the X-ray co-crystal structure of this complex, but provide additional information based on shifts in dynamics that are observed just outside the interface. </div><div><br></div><div>- These dynamic changes occur specifically in regions that are the primary targets of neutralizing antibodies that target spike protein, suggesting that the neutralization mechanism may result from suppression of dynamic shifts in the spike Receptor Binding Domain (RBD) that are necessary for favorable binding thermodynamics in the spike / ACE2 interaction.</div>

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Mutation-induced Changes in the Receptor-binding Interface of the SARS-CoV-2 Delta Variant B.1.617.2 and Implications for Immune Evasion

10.1101/2021.07.17.452576 ◽

2021 ◽

Author(s):

Prabin Baral ◽

Nisha Bhattarai ◽

Md Lokman Hossen ◽

Vitalii Stebliankin ◽

Bernard Gerstman ◽

...

Keyword(s):

Receptor Binding ◽

Immune Evasion ◽

Genetic Variants ◽

Neutralizing Antibodies ◽

Structural Changes ◽

Spike Protein ◽

Sharp Rise ◽

Stable Conformation ◽

Binding Interface ◽

Induced Changes

While the vaccination efforts against SARS-CoV-2 infections are ongoing worldwide, new genetic variants of the virus are emerging and spreading. Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant's receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding beta-loop-beta motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.

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