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1.
MAbs ; 14(1): 2005507, 2022.
Article in English | MEDLINE | ID: covidwho-1585297

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Antibody Affinity/immunology , Antibody Specificity/immunology , CHO Cells , COVID-19/prevention & control , COVID-19/virology , Chromatography, High Pressure Liquid/methods , Circular Dichroism , Clone Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/therapeutic use , Isoelectric Point , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism
2.
Front Immunol ; 12: 766821, 2021.
Article in English | MEDLINE | ID: covidwho-1581335

ABSTRACT

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and spread around the world, antibodies and vaccines to confer broad and potent neutralizing activity are urgently needed. Through the isolation and characterization of monoclonal antibodies (mAbs) from individuals infected with SARS-CoV-2, we identified one antibody, P36-5D2, capable of neutralizing the major SARS-CoV-2 variants of concern. Crystal and electron cryo-microscopy (cryo-EM) structure analyses revealed that P36-5D2 targeted to a conserved epitope on the receptor-binding domain of the spike protein, withstanding the three key mutations-K417N, E484K, and N501Y-found in the variants that are responsible for escape from many potent neutralizing mAbs, including some already approved for emergency use authorization (EUA). A single intraperitoneal (IP) injection of P36-5D2 as a prophylactic treatment completely protected animals from challenge of infectious SARS-CoV-2 Alpha and Beta. Treated animals manifested normal body weight and were devoid of infection-associated death up to 14 days. A substantial decrease of the infectious virus in the lungs and brain, as well as reduced lung pathology, was found in these animals compared to the controls. Thus, P36-5D2 represents a new and desirable human antibody against the current and emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , HEK293 Cells , Humans , Immunization, Passive , Mice
3.
Rev Med Virol ; 31(6): e2231, 2021 11.
Article in English | MEDLINE | ID: covidwho-1574317

ABSTRACT

The Spike protein is the target of both antibody-based therapeutics (convalescent plasma, polyclonal serum, monoclonal antibodies) and vaccines. Mutations in Spike could affect efficacy of those treatments. Hence, monitoring of mutations is necessary to forecast and readapt the inventory of therapeutics. Different phylogenetic nomenclatures have been used for the currently circulating SARS-CoV-2 clades. The Spike protein has different hotspots of mutation and deletion, the most dangerous for immune escape being the ones within the receptor binding domain (RBD), such as K417N/T, N439K, L452R, Y453F, S477N, E484K, and N501Y. Convergent evolution has led to different combinations of mutations among different clades. In this review we focus on the main variants of concern, that is, the so-called UK (B.1.1.7), South African (B.1.351) and Brazilian (P.1) strains.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , COVID-19/therapy , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/chemistry , Antibodies, Viral/metabolism , Antibodies, Viral/therapeutic use , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Gene Expression , Humans , Immune Evasion , Immunization, Passive/methods , Mutation , Phylogeny , Protein Binding , Risk Assessment , SARS-CoV-2/classification , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , South Africa/epidemiology , Spike Glycoprotein, Coronavirus/immunology , United Kingdom/epidemiology
5.
Cell Rep ; 37(3): 109869, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1517084

ABSTRACT

The dramatically expanding coronavirus disease 2019 (COVID-19) needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several receptor binding domain (RBD)-specific Nbs isolated from an Nb library derived from an alpaca immunized with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S); among them, three Nbs exhibit picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and circulating SARS-CoV-2 variants. To improve their efficacy, various configurations of Nbs are engineered. Nb15-NbH-Nb15, a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibits single-digit ng/ml neutralization potency against the wild-type and Delta variants of SARS-CoV-2 with a long half-life in vivo. In addition, we show that intranasal administration of Nb15-NbH-Nb15 provides effective protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both the prevention and treatment of SARS-CoV-2 through respiratory administration.


Subject(s)
Administration, Intranasal , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Bispecific/immunology , COVID-19/immunology , SARS-CoV-2 , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing , Antibodies, Viral/immunology , Camelids, New World , Epitopes/chemistry , Female , Humans , Kinetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neutralization Tests , Protein Binding , Protein Domains , Protein Engineering/methods , Serum Albumin, Human/chemistry , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus/immunology
6.
Nat Commun ; 12(1): 6304, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1500462

ABSTRACT

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibodies, Viral/administration & dosage , Antibodies, Viral/chemistry , Binding Sites , COVID-19/pathology , COVID-19/virology , Epitopes , Humans , Mice , Mice, Transgenic , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Load/drug effects , Weight Loss/drug effects
7.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1493344

ABSTRACT

Here, we expressed two neutralizing monoclonal antibodies (Abs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; H4 and B38) in three formats: IgG1, IgA1 monomers (m), and IgA1 dimers (d) in glycoengineered Nicotiana benthamiana plants. All six Ab variants assembled properly and exhibited a largely homogeneous glycosylation profile. Despite modest variation in antigen binding between Ab formats, SARS-CoV-2 neutralization (NT) potency significantly increased in the following manner: IgG1 < IgA1-m < IgA1-d, with an up to 240-fold NT increase of dimers compared to corresponding monomers. Our results underscore that both IgA's structural features and multivalency positively impact NT potency. In addition, they emphasize the versatile use of plants for the rapid expression of complex human proteins.


Subject(s)
Antibodies, Monoclonal/chemistry , COVID-19/virology , Immunoglobulin A/chemistry , Immunoglobulin G/chemistry , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Neutralization Tests , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
9.
Cell Rep ; 37(4): 109881, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1458602

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has necessitated the rapid development of antibody-based therapies and vaccines as countermeasures. Here, we use cryoelectron microscopy (cryo-EM) to characterize two protective anti-SARS-CoV-2 murine monoclonal antibodies (mAbs) in complex with the spike protein, revealing similarities between epitopes targeted by human and murine B cells. The more neutralizing mAb, 2B04, binds the receptor-binding motif (RBM) of the receptor-binding domain (RBD) and competes with angiotensin-converting enzyme 2 (ACE2). By contrast, 2H04 binds adjacent to the RBM and does not compete for ACE2 binding. Naturally occurring sequence variants of SARS-CoV-2 and corresponding neutralization escape variants selected in vitro map to our structurally defined epitopes, suggesting that SARS-CoV-2 might evade therapeutic antibodies with a limited set of mutations, underscoring the importance of combination mAb therapeutics. Finally, we show that 2B04 neutralizes SARS-CoV-2 infection by preventing ACE2 engagement, whereas 2H04 reduces host cell attachment without directly disrupting ACE2-RBM interactions, providing distinct inhibitory mechanisms used by RBD-specific mAbs.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Cryoelectron Microscopy , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Humans , Mice , Protein Interaction Domains and Motifs/immunology , Protein Structure, Quaternary , Spike Glycoprotein, Coronavirus/chemistry
10.
Gigascience ; 10(9)2021 09 28.
Article in English | MEDLINE | ID: covidwho-1443047

ABSTRACT

BACKGROUND: B-cell immunoglobulin repertoires with paired heavy and light chain can be determined by means of 10X single-cell V(D)J sequencing. Precise and quick analysis of 10X single-cell immunoglobulin repertoires remains a challenge owing to the high diversity of immunoglobulin repertoires and a lack of specialized software that can analyze such diverse data. FINDINGS: In this study, specialized software for 10X single-cell immunoglobulin repertoire analysis was developed. SCIGA (Single-Cell Immunoglobulin Repertoire Analysis) is an easy-to-use pipeline that performs read trimming, immunoglobulin sequence assembly and annotation, heavy and light chain pairing, statistical analysis, visualization, and multiple sample integration analysis, which is all achieved by using a 1-line command. Then SCIGA was used to profile the single-cell immunoglobulin repertoires of 9 patients with coronavirus disease 2019 (COVID-19). Four neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified from these repertoires. CONCLUSIONS: SCIGA provides a complete and quick analysis for 10X single-cell V(D)J sequencing datasets. It can help researchers to interpret B-cell immunoglobulin repertoires with paired heavy and light chain.


Subject(s)
Immunoglobulins/metabolism , Single-Cell Analysis/methods , Software , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , COVID-19/pathology , COVID-19/virology , Humans , Immunoglobulins/chemistry , Immunoglobulins/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification
11.
Cell Rep ; 37(1): 109771, 2021 10 05.
Article in English | MEDLINE | ID: covidwho-1439919

ABSTRACT

Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Aged , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Binding Sites , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/ultrastructure , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Light Chains/ultrastructure , Male , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
12.
Nat Microbiol ; 6(10): 1233-1244, 2021 10.
Article in English | MEDLINE | ID: covidwho-1434113

ABSTRACT

Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , SARS-CoV-2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antigenic Variation , Binding Sites , COVID-19/immunology , COVID-19/virology , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Humans , Mutation , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
13.
ACS Appl Mater Interfaces ; 13(36): 43696-43707, 2021 Sep 15.
Article in English | MEDLINE | ID: covidwho-1392772

ABSTRACT

Graphene is a two-dimensional semiconducting material whose application for diagnostics has been a real game-changer in terms of sensitivity and response time, variables of paramount importance to stop the COVID-19 spreading. Nevertheless, strategies for the modification of docking recognition and antifouling elements to obtain covalent-like stability without the disruption of the graphene band structure are still needed. In this work, we conducted surface engineering of graphene through heterofunctional supramolecular-covalent scaffolds based on vinylsulfonated-polyamines (PA-VS). In these scaffolds, one side binds graphene through multivalent π-π interactions with pyrene groups, and the other side presents vinylsulfonated pending groups that can be used for covalent binding. The construction of PA-VS scaffolds was demonstrated by spectroscopic ellipsometry, Raman spectroscopy, and contact angle measurements. The covalent binding of -SH, -NH2, or -OH groups was confirmed, and it evidenced great chemical versatility. After field-effect studies, we found that the PA-VS-based scaffolds do not disrupt the semiconducting properties of graphene. Moreover, the scaffolds were covalently modified with poly(ethylene glycol) (PEG), which improved the resistance to nonspecific proteins by almost 7-fold compared to the widely used PEG-monopyrene approach. The attachment of recognition elements to PA-VS was optimized for concanavalin A (ConA), a model lectin with a high affinity to glycans. Lastly, the platform was implemented for the rapid, sensitive, and regenerable recognition of SARS-CoV-2 spike protein and human ferritin in lab-made samples. Those two are the target molecules of major importance for the rapid detection and monitoring of COVID-19-positive patients. For that purpose, monoclonal antibodies (mAbs) were bound to the scaffolds, resulting in a surface coverage of 436 ± 30 ng/cm2. KD affinity constants of 48.4 and 2.54 nM were obtained by surface plasmon resonance (SPR) spectroscopy for SARS-CoV-2 spike protein and human ferritin binding on these supramolecular scaffolds, respectively.


Subject(s)
Biomarkers/analysis , COVID-19/diagnosis , Graphite/chemistry , Immunoassay/methods , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Ethylenes/chemistry , Ferritins/immunology , Ferritins/metabolism , Humans , Point-of-Care Systems , Polyamines/chemistry , Polyethylene Glycols/chemistry , Pyrenes/chemistry , Quantum Theory , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Semiconductors , Spike Glycoprotein, Coronavirus/immunology , Sulfonic Acids/chemistry , Surface Plasmon Resonance
14.
Science ; 372(6546): 1108-1112, 2021 06 04.
Article in English | MEDLINE | ID: covidwho-1388437

ABSTRACT

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an amino (N)-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multidonor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/chemistry , Antibodies, Viral/blood , Antibodies, Viral/chemistry , Antibody Affinity , COVID-19/prevention & control , Epitopes/immunology , Humans , Immune Evasion , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Mice , Mice, Inbred BALB C , Mutation , Protein Domains , Proteomics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
15.
Nat Commun ; 12(1): 250, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-1387324

ABSTRACT

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Disease Models, Animal , Epitopes , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
16.
Immunity ; 54(10): 2399-2416.e6, 2021 10 12.
Article in English | MEDLINE | ID: covidwho-1364126

ABSTRACT

With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Amino Acid Motifs , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Epitopes/chemistry , Epitopes/metabolism , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/metabolism , Mice , Neutralization Tests , Protein Domains , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
17.
Cell Rep ; 36(10): 109679, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1363916

ABSTRACT

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/chemistry , Antibody Affinity , COVID-19/therapy , COVID-19/virology , Epitopes/genetics , Epitopes/immunology , Humans , Immunization, Passive , Mice , Mice, Transgenic , Models, Molecular , Neutralization Tests , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Treatment Outcome
18.
Nat Commun ; 12(1): 4676, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1340999

ABSTRACT

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.


Subject(s)
Broadly Neutralizing Antibodies/immunology , Epitopes/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Binding Sites , Broadly Neutralizing Antibodies/chemistry , Broadly Neutralizing Antibodies/classification , Broadly Neutralizing Antibodies/metabolism , COVID-19/drug therapy , COVID-19/prevention & control , Epitopes/chemistry , Epitopes/metabolism , Humans , Models, Molecular , Mutation , Protein Binding , SARS-CoV-2/genetics , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/classification , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
19.
Nature ; 597(7874): 103-108, 2021 09.
Article in English | MEDLINE | ID: covidwho-1316713

ABSTRACT

The recent emergence of SARS-CoV-2 variants of concern1-10 and the recurrent spillovers of coronaviruses11,12 into the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here we describe a human monoclonal antibody designated S2X259, which recognizes a highly conserved cryptic epitope of the receptor-binding domain and cross-reacts with spikes from all clades of sarbecovirus. S2X259 broadly neutralizes spike-mediated cell entry of SARS-CoV-2, including variants of concern (B.1.1.7, B.1.351, P.1, and B.1.427/B.1.429), as well as a wide spectrum of human and potentially zoonotic sarbecoviruses through inhibition of angiotensin-converting enzyme 2 (ACE2) binding to the receptor-binding domain. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2X259 possesses an escape profile that is limited to a single substitution, G504D. We show that prophylactic and therapeutic administration of S2X259 protects Syrian hamsters (Mesocricetus auratus) against challenge with the prototypic SARS-CoV-2 and the B.1.351 variant of concern, which suggests that this monoclonal antibody is a promising candidate for the prevention and treatment of emergent variants and zoonotic infections. Our data reveal a key antigenic site that is targeted by broadly neutralizing antibodies and will guide the design of vaccines that are effective against all sarbecoviruses.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , Broadly Neutralizing Antibodies/therapeutic use , COVID-19/prevention & control , SARS-CoV-2/classification , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , Broadly Neutralizing Antibodies/chemistry , COVID-19/immunology , COVID-19/virology , Cross Reactions/immunology , Disease Models, Animal , Female , Humans , Immune Evasion/genetics , Immune Evasion/immunology , Mesocricetus/immunology , Mesocricetus/virology , Mutation , Neutralization Tests , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Viral Zoonoses/immunology , Viral Zoonoses/prevention & control , Viral Zoonoses/virology
20.
Nature ; 597(7874): 97-102, 2021 09.
Article in English | MEDLINE | ID: covidwho-1309448

ABSTRACT

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse sarbecoviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.


Subject(s)
Broadly Neutralizing Antibodies/immunology , COVID-19/virology , Cross Reactions/immunology , Immune Evasion , SARS-CoV-2/classification , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibody Affinity , Broadly Neutralizing Antibodies/chemistry , COVID-19/drug therapy , COVID-19/immunology , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Cell Line , Cricetinae , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immune Evasion/genetics , Immune Evasion/immunology , Male , Mesocricetus , Middle Aged , Models, Molecular , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccinology
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