A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants

The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19.

Download Full-text

Isolation of a human monoclonal antibody specific for the receptor binding domain of SARS-CoV-2 using a competitive phage biopanning strategy

Antibody Therapeutics ◽

10.1093/abt/tbaa008 ◽

2020 ◽

Vol 3 (2) ◽

pp. 95-100 ◽

Cited By ~ 8

Author(s):

Xin Zeng ◽

Lingfang Li ◽

Jing Lin ◽

Xinlei Li ◽

Bin Liu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Magnetic Beads ◽

Human Monoclonal Antibody ◽

Binding Domain ◽

Receptor Binding Domain ◽

Antibody Library ◽

Synthetic Antibody ◽

Blocking Antibodies

Abstract The infection of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 200 000 deaths, but no vaccine or therapeutic monoclonal antibody is currently available. SARS-CoV-2 relies on its spike protein, in particular the receptor-binding domain (RBD), to bind human cell receptor angiotensin-converting enzyme 2 (ACE2) for viral entry, and thus targeting RBD holds the promise for preventing SARS-CoV-2 infection. In this work, a competitive biopanning strategy of a phage display antibody library was applied to screen blocking antibodies against RBD. High-affinity antibodies were enriched after the first round using a standard panning process in which RBD-His was immobilized as a bait. At the next two rounds, immobilized ACE2-Fc and free RBD-His were mixed with the enriched phage antibodies. Antibodies binding to RBD at epitopes different from ACE2-binding site were captured by the immobilized ACE2-Fc, forming a “sandwich” complex. Only antibodies competed with ACE2 can bind to the free RBD-His in the supernatant and be subsequently separated by the nickel-nitrilotriacetic acid magnetic beads. rRBD-15 from the competitive biopanning of our synthetic antibody library, Lib AB1, was produced as the full-length IgG1 format. It was proved to competitively block the binding of RBD to ACE2 and potently inhibit SARS-CoV-2 pseudovirus infection with IC50 values of 12 nM. Nevertheless, rRBD-16 from the standard biopanning can only bind to RBD in vitro, but not have the blocking or neutralization activity. Our strategy can efficiently isolate the blocking antibodies of RBD, and it would speed up the discovery of neutralizing antibodies against SARS-CoV-2.

Download Full-text

Epistasis at the SARS-CoV-2 RBD Interface and the Propitiously Boring Implications for Vaccine Escape

10.1101/2021.08.30.458225 ◽

2021 ◽

Cited By ~ 1

Author(s):

Nash D. Rochman ◽

Guilhem Faure ◽

Yuri I. Wolf ◽

Peter L. Freddolino ◽

Feng Zhang ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Wild Type ◽

Protein Structure Modeling ◽

Escape Mutants ◽

Small Set ◽

Global Concern ◽

The Impact

AbstractAt the time of this writing, August 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlap with the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple mutations in the RBD have rose to dominance. Non-additive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape is crucial. Epistasis can substantially increase the risk of vaccine escape and cannot be completely characterized through the study of the wild type (WT) alone. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the WT, Gamma (417T, 484K, 501Y), and Delta variants (452R, 478K). Overall, epistasis at the RBD surface appears to be limited and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2, whereas in the Gamma variant, epistasis more substantially destabilizes NAb interaction. These results suggest that the repertoire of potential escape mutations for the Delta variant is not substantially different from that of the WT, whereas Gamma poses a moderately greater risk for enhanced vaccine escape. Thus, the modest ensemble of mutations relative to the WT shown to reduce vaccine efficacy might constitute the majority of all possible escape mutations.SignificancePotential emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild type SARS-CoV-2 virus as well as the Gamma and Delta variants. The results indicate that emergence of escape mutants is somewhat less likely for the Delta variant than for the wild type and moderately more likely for the Gamma variant. We conclude that the small set of escape-enhancing mutations already identified for the wild type is likely to include the majority of all possible mutations with this effect, a welcome finding.

Download Full-text

A Recombinant Protein SARS-CoV-2 Candidate Vaccine Elicits High-titer Neutralizing Antibodies in Macaques.

10.21203/rs.3.rs-137857/v1 ◽

2021 ◽

Author(s):

Gary Baisa ◽

David Rancour ◽

Keith Mansfield ◽

Monika Burns ◽

Lori Martin ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

High Titer ◽

Binding Domain ◽

Pseudotyped Virus ◽

Receptor Binding Domain ◽

Binding Assays ◽

Neutralizing Activity ◽

Terminal Domain

Abstract BackgroundVaccines that generate robust and long-lived protective immunity against SARS-CoV-2 infection are urgently required. MethodsWe assessed the potential of vaccine candidates based on the SARS-CoV-2 spike in cynomolgus macaques (M. fascicularis) by examining their ability to generate spike binding antibodies with neutralizing activity. Antigens were derived from two distinct regions of the spike S1 subunit, either the N-terminal domain or an extended C-terminal domain containing the receptor-binding domain and were fused to the human IgG1 Fc domain. Three groups of 2 animals each were immunized with either antigen, alone or in combination. The development of antibody responses was evaluated through 20 weeks post-immunization. ResultsA robust IgG response to the spike protein was detected as early as 2 weeks after immunization with either protein and maintained for over 20 weeks. Sera from animals immunized with antigens derived from the RBD were able to prevent binding of soluble spike proteins to the ACE2 receptor, shown by in vitro binding assays, while sera from animals immunized with the N-terminal domain alone lacked this activity. Crucially, sera from animals immunized with the extended receptor binding domain but not the N-terminal domain had potent neutralizing activity against SARS-CoV-2 pseudotyped virus, with titers in excess of 10,000, greatly exceeding that typically found in convalescent humans. Neutralizing activity persisted for more than 20 weeks. ConclusionsThese data support the utility of spike subunit-based antigens as a vaccine for use in humans.

Download Full-text

Potent SARS-CoV-2 Neutralizing Antibodies Directed Against Spike N-Terminal Domain Target a Single Supersite

10.1101/2021.01.10.426120 ◽

2021 ◽

Cited By ~ 3

Author(s):

Gabriele Cerutti ◽

Yicheng Guo ◽

Tongqing Zhou ◽

Jason Gorman ◽

Myungjin Lee ◽

...

Keyword(s):

Free Surface ◽

Crystal Structures ◽

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Viral Membrane ◽

Terminal Domain ◽

Flexible Loops

SummaryNumerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycans N17, N74, N122, and N149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.

Download Full-text

The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines

10.1101/2021.08.22.457114 ◽

2021 ◽

Cited By ~ 2

Author(s):

Yafei Liu ◽

Noriko Arase ◽

Jun-ichi Kishikawa ◽

Mika Hirose ◽

Songling Li ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Binding Domain ◽

Receptor Binding Domain ◽

Wild Type ◽

Effective Protection ◽

Neutralizing Activity ◽

Terminal Domain ◽

Complete Resistance

AbstractmRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

Download Full-text

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.

Download Full-text

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Science ◽

10.1126/science.abg9175 ◽

2021 ◽

pp. eabg9175 ◽

Cited By ~ 6

Author(s):

Leonidas Stamatatos ◽

Julie Czartoski ◽

Yu-Hsin Wan ◽

Leah J. Homad ◽

Vanessa Rubin ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Binding Domain ◽

Receptor Binding Domain ◽

Previous Infection

Emerging SARS-CoV-2 variants have raised concerns about resistance to neutralizing antibodies elicited by previous infection or vaccination. We examined whether sera from recovered and naïve donors collected prior to, and following immunizations with existing mRNA vaccines, could neutralize the Wuhan-Hu-1 and B.1.351 variants. Pre-vaccination sera from recovered donors neutralized Wuhan-Hu-1 and sporadically neutralized B.1.351, but a single immunization boosted neutralizing titers against all variants and SARS-CoV-1 by up to 1000-fold. Neutralization was due to antibodies targeting the receptor binding domain and was not boosted by a second immunization. Immunization of naïve donors also elicited cross-neutralizing responses, but at lower titers. Our study highlights the importance of vaccinating both uninfected and previously infected persons to elicit cross-variant neutralizing antibodies.

Download Full-text

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

Nature Communications ◽

10.1038/s41467-020-20654-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Tiong Kit Tan ◽

Pramila Rijal ◽

Rolle Rahikainen ◽

Anthony H. Keeble ◽

Lisa Schimanski ◽

...

Keyword(s):

Antibody Response ◽

Receptor Binding ◽

Binding Domain ◽

Cold Chain ◽

Receptor Binding Domain ◽

Cell Infection ◽

Protein Receptor ◽

Virus Like Particle ◽

Human Sera ◽

Escape Mutants

AbstractThere is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic.

Download Full-text