Computational redesign of Fab CC12.3 with substantially better predicted binding affinity to SARS-CoV-2 than human ACE2 receptor

AbstractSARS-CoV-2 is responsible for COVID-19 pandemic, causing large numbers of cases and deaths. It initiates entry into human cells by binding to the peptidase domain of angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain of S1 subunit of spike protein (SARS-CoV-2-RBD). Employing neutralizing antibodies to prevent binding between SARS-CoV-2-RBD and ACE2 is an effective COVID-19 therapeutic solution. Previous studies found that CC12.3 is a highly potent neutralizing antibody that was isolated from a SARS-CoV-2 infected patient, and its Fab fragment (Fab CC12.3) bound to SARS-CoV-2-RBD with comparable binding affinity to ACE2. To enhance its binding affinity, we employed computational protein design to redesign all CDRs of Fab CC12.3 and molecular dynamics (MD) to validate their predicted binding affinities by the MM-GBSA method. MD results show that the predicted binding affinities of the three best designed Fabs CC12.3 (CC12.3-D02, CC12.3-D05, and CC12.3-D08) are better than those of Fab CC12.3 and ACE2. Additionally, our results suggest that enhanced binding affinities of CC12.3-D02, CC12.3-D05, and CC12.3-D08 are caused by increased SARS-CoV-2-RBD binding interactions of CDRs L1 and L3. This study redesigned neutralizing antibodies with better predicted binding affinities to SARS-CoV-2-RBD than Fab CC12.3 and ACE2. They are promising candidates as neutralizing antibodies against SARS-CoV-2.

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Computational design of SARS-CoV-2 peptide binders with better predicted binding affinities than human ACE2 receptor

Scientific Reports ◽

10.1038/s41598-021-94873-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Thassanai Sitthiyotha ◽

Surasak Chunsrivirot

Keyword(s):

Experimental Study ◽

Protein Design ◽

Computational Design ◽

Binding Affinities ◽

Receptor Binding Domain ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Lower Affinity ◽

Previous Experimental Study ◽

Better Than

AbstractSARS-CoV-2 is coronavirus causing COVID-19 pandemic. To enter human cells, receptor binding domain of S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) binds to peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptor. Employing peptides to inhibit binding between SARS-CoV-2-RBD and ACE2-PD is a therapeutic solution for COVID-19. Previous experimental study found that 23-mer peptide (SBP1) bound to SARS-CoV-2-RBD with lower affinity than ACE2. To increase SBP1 affinity, our previous study used residues 21–45 of α1 helix of ACE2-PD (SPB25) to design peptides with predicted affinity better than SBP1 and SPB25 by increasing interactions of residues that do not form favorable interactions with SARS-CoV-2-RBD. To design SPB25 with better affinity than ACE2, we employed computational protein design to increase interactions of residues reported to form favorable interactions with SARS-CoV-2-RBD and combine newly designed mutations with the best single mutations from our previous study. Molecular dynamics show that predicted binding affinities of three peptides (SPB25Q22R, SPB25F8R/K11W/L25R and SPB25F8R/K11F/Q22R/L25R) are better than ACE2. Moreover, their predicted stabilities may be slightly higher than SBP1 as suggested by their helicities. This study developed an approach to design SARS-CoV-2 peptide binders with predicted binding affinities better than ACE2. These designed peptides are promising candidates as SARS-CoV-2 inhibitors.

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The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in non-human primates

Science Translational Medicine ◽

10.1126/scitranslmed.abf1906 ◽

2021 ◽

pp. eabf1906

Author(s):

Bryan E. Jones ◽

Patricia L. Brown-Augsburger ◽

Kizzmekia S. Corbett ◽

Kathryn Westendorf ◽

Julian Davies ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Functional Characterization ◽

Neutralizing Antibody ◽

Therapeutic Agents ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Cynomolgus Monkeys ◽

Binding Inhibition ◽

Vaccination Campaigns

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

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Development of a Rapid Point-Of-Care Test that Measures Neutralizing Antibodies to SARS-CoV-2

10.1101/2020.12.15.20248264 ◽

2020 ◽

Author(s):

Douglas F. Lake ◽

Alexa J. Roeder ◽

Erin Kaleta ◽

Paniz Jasbi ◽

Sivakumar Periasamy ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Point Of Care ◽

Neutralizing Antibody ◽

Antibody Test ◽

Spike Protein ◽

Receptor Binding Domain ◽

Plasma Therapy ◽

Angiotensin Converting Enzyme 2 ◽

Point Of Care Test ◽

Protein Receptor

As increasing numbers of people recover from and are vaccinated against COVID-19, tests are needed to measure levels of protective, neutralizing antibodies longitudinally to help determine duration of immunity. We developed a lateral flow assay (LFA) that measures levels of neutralizing antibodies in plasma, serum or whole blood. The LFA is based on the principle that neutralizing antibodies inhibit binding of the spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2). The test classifies high levels of neutralizing antibodies in sera that were titered using authentic SARS-CoV-2 and pseudotype neutralization assays with an accuracy of 98%. Sera obtained from patients with seasonal coronavirus did not prevent RBD from binding to ACE2. As a demonstration for convalescent plasma therapy, we measured conversion of non-immune plasma into strongly neutralizing plasma. This is the first report of a neutralizing antibody test that is rapid, highly portable and relatively inexpensive that might be useful in assessing COVID-19 vaccine immunity.

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Comprehensive Deep Mutational Scanning Reveals the Immune-Escaping Hotspots of SARS-CoV-2 Receptor-Binding Domain Targeting Neutralizing Antibodies

Frontiers in Microbiology ◽

10.3389/fmicb.2021.698365 ◽

2021 ◽

Vol 12 ◽

Author(s):

Keng-Chang Tsai ◽

Yu-Ching Lee ◽

Tien-Sheng Tseng

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Human Immune System ◽

Angiotensin Converting Enzyme 2 ◽

Study Results ◽

Rapid Spread ◽

Care Systems ◽

Computational Analyses

The rapid spread of SARS-CoV-2 has caused the COVID-19 pandemic, resulting in the collapse of medical care systems and economic depression worldwide. To combat COVID-19, neutralizing antibodies have been investigated and developed. However, the evolutions (mutations) of the receptor-binding domain (RBD) of SARS-CoV-2 enable escape from neutralization by these antibodies, further impairing recognition by the human immune system. Thus, it is critical to investigate and predict the putative mutations of RBD that escape neutralizing immune responses. Here, we employed computational analyses to comprehensively investigate the mutational effects of RBD on binding to neutralizing antibodies and angiotensin-converting enzyme 2 (ACE2) and demonstrated that the RBD residues K417, L452, L455, F456, E484, G485, F486, F490, Q493, and S494 were consistent with clinically emerging variants or experimental observations of attenuated neutralizations. We also revealed common hotspots, Y449, L455, and Y489, that exerted comparable destabilizing effects on binding to both ACE2 and neutralizing antibodies. Our results provide valuable information on the putative effects of RBD variants on interactions with neutralizing antibodies. These findings provide insights into possible evolutionary hotspots that can escape recognition by these antibodies. In addition, our study results will benefit the development and design of vaccines and antibodies to combat the newly emerging variants of SARS-CoV-2.

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Cross-neutralization antibodies against SARS-CoV-2 and RBD mutations from convalescent patient antibody libraries

10.1101/2020.06.06.137513 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yan Lou ◽

Wenxiang Zhao ◽

Haitao Wei ◽

Min Chu ◽

Ruihua Chao ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Viral Infections ◽

Therapeutic Agents ◽

Therapeutic Interventions ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Human Antibodies ◽

Global Pandemic ◽

Antibody Libraries ◽

Cross Neutralization

AbstractThe emergence of coronavirus disease 2019 (COVID-19) pandemic led to an urgent need to develop therapeutic interventions. Among them, neutralizing antibodies play crucial roles for preventing viral infections and contribute to resolution of infection. Here, we describe the generation of antibody libraries from 17 different COVID-19 recovered patients and screening of neutralizing antibodies to SARS-CoV-2. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Then the positive clones were sequenced and reconstituted into whole human IgG for epitope binning assays. After that, all 19 IgG were classified into 6 different epitope groups or Bins. Although all these antibodies were shown to have ability to bind RBD, the antibodies in Bin2 have more superiority to inhibit the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 can also strongly bind with mutant RBDs (W463R, R408I, N354D, V367F and N354D/D364Y) derived from SARS-CoV-2 strain with increased infectivity, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, these neutralizing antibodies strongly restrict the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, these antibodies can even block the entry of live SARS-CoV-2 into cells at only 12.5 nM. These results suggest that these neutralizing human antibodies from the patient-derived antibody libraries have the potential to become therapeutic agents against SARS-CoV-2 and its mutants in this global pandemic.

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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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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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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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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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