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1.
PLoS Comput Biol ; 17(12): e1009675, 2021 12.
Article in English | MEDLINE | ID: covidwho-1619980

ABSTRACT

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.


Subject(s)
Antigens, Viral/chemistry , COVID-19/immunology , COVID-19/virology , Epitopes, B-Lymphocyte/chemistry , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/genetics , Antigen-Antibody Reactions/genetics , Antigen-Antibody Reactions/immunology , Computational Biology , Coronavirus/chemistry , Coronavirus/genetics , Coronavirus/immunology , Databases, Chemical , Epitope Mapping , Epitopes, B-Lymphocyte/genetics , Humans , Mice , Models, Molecular , Pandemics , SARS-CoV-2/genetics , Single-Domain Antibodies/immunology
2.
Nat Commun ; 12(1): 7325, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1585854

ABSTRACT

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Crystallography, X-Ray , Receptors, Antigen/chemistry , Receptors, Antigen/immunology , Sharks/immunology , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Epitopes , Mutation , Phylogeny , Protein Binding , SARS-CoV-2 , Sequence Alignment , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus/immunology
3.
MAbs ; 14(1): 2002236, 2022.
Article in English | MEDLINE | ID: covidwho-1585298

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques , Immunoglobulin G/immunology , Peptide Library , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Binding Sites, Antibody , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Epitopes , Female , Host-Pathogen Interactions , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Mesocricetus , SARS-CoV-2/pathogenicity , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacology , Vero Cells
4.
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
5.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Article in English | MEDLINE | ID: covidwho-1470027

ABSTRACT

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , COVID-19/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Antigen-Presenting Cells/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , COVID-19 Vaccines/administration & dosage , Camelids, New World/immunology , Female , Histocompatibility Antigens Class II/immunology , Humans , Immunity, Cellular , Immunity, Humoral , Immunization, Secondary , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Pandemics/prevention & control , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/genetics , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
PLoS Pathog ; 17(10): e1009542, 2021 10.
Article in English | MEDLINE | ID: covidwho-1468184

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.


Subject(s)
Antibodies, Viral/administration & dosage , Antiviral Agents/administration & dosage , COVID-19 , Single-Domain Antibodies/administration & dosage , Virus Attachment/drug effects , Administration, Intranasal , Animals , Chlorocebus aethiops , Cricetinae , Disease Models, Animal , Humans , Mesocricetus , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
7.
FASEB J ; 35(11): e21970, 2021 11.
Article in English | MEDLINE | ID: covidwho-1462503

ABSTRACT

Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin-converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , COVID-19/immunology , COVID-19/prevention & control , Chlorocebus aethiops , Humans , Protein Binding , Sharks/immunology , Vero Cells
8.
Proc Natl Acad Sci U S A ; 118(41)2021 10 12.
Article in English | MEDLINE | ID: covidwho-1462069

ABSTRACT

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody-target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Šoverall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Šresolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.


Subject(s)
Cryoelectron Microscopy/methods , SARS-CoV-2/metabolism , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites/immunology , COVID-19/virology , HEK293 Cells , Humans , Models, Molecular , Protein Binding , Protein Domains , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/ultrastructure
9.
J Chem Inf Model ; 61(10): 5152-5160, 2021 10 25.
Article in English | MEDLINE | ID: covidwho-1440449

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human cells upon binding of its spike (S) glycoproteins to ACE2 receptors. Several nanobodies neutralize SARS-CoV-2 infection by binding to the receptor-binding domain (RBD) of the S protein, but how their binding antagonizes S-ACE2 interactions is not well understood. Here, we identified interactions between the RBD and nanobodies H11-H4, H11-D4, and Ty1 by performing all-atom molecular dynamics simulations. H11-H4 and H11-D4 can bind to RBD without overlapping with ACE2. H11-H4, and to a lesser extent H11-D4, binding dislocates ACE2 from its binding site due to electrostatic repulsion. In comparison, Ty1 overlaps with ACE2 on RBD and has a similar binding strength to ACE2. Mutations in the Alpha variant of SARS-CoV-2 had a minor effect in RBD binding strengths of ACE2 and nanobodies, but reduced the ability of H11-H4 and H11-D4 to dislocate ACE2 from RBD. In comparison, the Beta variant weakened the RBD binding strengths of H11-H4 and H11-D4, which were less effective to dislocate ACE2 binding. Unexpectedly, mutations in Beta strengthened Ty1 binding to RBD, suggesting that this nanobody may be more effective to neutralize the Beta variant of SARS-CoV-2.


Subject(s)
COVID-19 , Single-Domain Antibodies , Binding Sites , Humans , Protein Binding , SARS-CoV-2
10.
Nat Commun ; 12(1): 5469, 2021 09 22.
Article in English | MEDLINE | ID: covidwho-1434103

ABSTRACT

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Subject(s)
Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , Single-Domain Antibodies/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Administration, Intranasal , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Cryoelectron Microscopy , Crystallography, X-Ray , Disease Models, Animal , Dose-Response Relationship, Immunologic , Epitopes/chemistry , Epitopes/metabolism , Female , Male , Mesocricetus , Neutralization Tests , SARS-CoV-2/drug effects , Single-Domain Antibodies/administration & dosage , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
11.
Nat Commun ; 12(1): 5506, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1428815

ABSTRACT

Antibody engineering technologies face increasing demands for speed, reliability and scale. We develop CeVICA, a cell-free nanobody engineering platform that uses ribosome display for in vitro selection of nanobodies from a library of 1011 randomized sequences. We apply CeVICA to engineer nanobodies against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein and identify >800 binder families using a computational pipeline based on CDR-directed clustering. Among 38 experimentally-tested families, 30 are true RBD binders and 11 inhibit SARS-CoV-2 pseudotyped virus infection. Affinity maturation and multivalency engineering increase nanobody binding affinity and yield a virus neutralizer with picomolar IC50. Furthermore, the capability of CeVICA for comprehensive binder prediction allows us to validate the fitness of our nanobody library. CeVICA offers an integrated solution for rapid generation of divergent synthetic nanobodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel nanobody engineering.


Subject(s)
Antibodies, Neutralizing/immunology , Protein Engineering , SARS-CoV-2/drug effects , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/pharmacology , Antibodies, Viral , COVID-19/drug therapy , Humans , Protein Binding , Reproducibility of Results , Single-Domain Antibodies/genetics , Spike Glycoprotein, Coronavirus
12.
J Biol Chem ; 297(4): 101202, 2021 10.
Article in English | MEDLINE | ID: covidwho-1428100

ABSTRACT

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.


Subject(s)
Antigen-Antibody Complex , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Crystallography, X-Ray , Humans , Protein Binding , Protein Domains , Protein Stability , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Sequence Alignment , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
13.
J Mol Graph Model ; 109: 108035, 2021 12.
Article in English | MEDLINE | ID: covidwho-1415578

ABSTRACT

The pandemic of the COVID-19 disease caused by SARS-CoV-2 has led to more than 200 million infections and over 4 million deaths worldwide. The progress in the developments of effective vaccines and neutralizing antibody therapeutics brings hopes to eliminate the threat of COVID-19. However, SARS-CoV-2 continues to mutate, and several new variants have been emerged. Among the various naturally-occurring mutations, the E484K mutation shared by many variants attracted serious concerns, which may potentially enhance the receptor binding affinity and reduce the immune response. In the present study, the molecular mechanism behind the impacts of E484K mutation on the binding affinity of the receptor-binding domain (RBD) with the receptor human angiotensin-converting enzyme 2 (hACE2) was investigated by using the molecular dynamics (MD) simulations combined with the molecular mechanics-generalized Born surface area (MMGBSA) method. Our results indicate that the E484K mutation results in more favorable electrostatic interactions compensating the burial of the charged and polar groups upon the binding of RBD with hACE2, which significantly improves the RBD-hACE2 binding affinity. Besides that, the E484K mutation also causes the conformational rearrangements of the loop region containing the mutant residue, which leads to tighter binding interface of RBD with hACE2 and formation of some new hydrogen bonds. The tighter binding interface and the new hydrogen bonds formation also contribute to the improved binding affinity of RBD to the receptor hACE2. In addition, six neutralizing antibodies and nanobodies complexed with RBD were selected to explore the effects of E484K mutation on the recognition of these antibodies to RBD. The simulation results show that the E484K mutation significantly reduces the binding affinities to RBD for most of the studied neutralizing antibodies/nanobodies, and the decrease in the binding affinities is mainly owing to the unfavorable electrostatic interactions caused by the mutation. Our studies revealed that the E484K mutation may improve the binding affinity between RBD and the receptor hACE2, implying more transmissibility of the E484K-containing variants, and weaken the binding affinities between RBD and the studied neutralizing antibodies/nanobodies, indicating reduced effectiveness of these antibodies/nanobodies. Our results provide valuable information for the effective vaccine development and antibody/nanobody drug design.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19 , Single-Domain Antibodies , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Neutralizing , Humans , Mutation , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
14.
Signal Transduct Target Ther ; 6(1): 202, 2021 05 22.
Article in English | MEDLINE | ID: covidwho-1387228
15.
Cell Chem Biol ; 28(9): 1379-1388.e7, 2021 09 16.
Article in English | MEDLINE | ID: covidwho-1385232

ABSTRACT

There is widespread interest in facile methods for generating potent neutralizing antibodies, nanobodies, and other affinity proteins against SARS-CoV-2 and related viruses to address current and future pandemics. While isolating antibodies from animals and humans are proven approaches, these methods are limited to the affinities, specificities, and functional activities of antibodies generated by the immune system. Here we report a surprisingly simple directed evolution method for generating nanobodies with high affinities and neutralization activities against SARS-CoV-2. We demonstrate that complementarity-determining region swapping between low-affinity lead nanobodies, which we discovered unintentionally but find is simple to implement systematically, results in matured nanobodies with unusually large increases in affinity. Importantly, the matured nanobodies potently neutralize both SARS-CoV-2 pseudovirus and live virus, and possess drug-like biophysical properties. We expect that our methods will improve in vitro nanobody discovery and accelerate the generation of potent neutralizing nanobodies against diverse coronaviruses.


Subject(s)
Antibodies, Neutralizing/genetics , Complementarity Determining Regions/genetics , Single-Domain Antibodies/genetics , Animals , Antibodies, Neutralizing/chemistry , Chlorocebus aethiops , Epitopes , HEK293 Cells , Humans , Mutagenesis , SARS-CoV-2 , Saccharomyces cerevisiae , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
16.
Front Immunol ; 12: 690742, 2021.
Article in English | MEDLINE | ID: covidwho-1389184

ABSTRACT

Since December 2019, the SARS-CoV-2 has erupted on a large scale worldwide and spread rapidly. Passive immunization of antibody-related molecules provides opportunities for prevention and treatment of high-risk patients and children. Nanobodies (Nbs) have many strong physical and chemical properties. They can be atomized, administered by inhalation, and can be directly applied to the infected site, with fast onset, high local drug concentration/high bioavailability, and high patient compliance (no needles). It has very attractive potential in the treatment of respiratory viruses. Rapid and low-cost development of Nbs targeting SARS-CoV-2 can quickly be achieved. Nbs against SARS-CoV-2 mutant strains also can be utilized quickly to prevent the virus from escaping. It provides important technical supports for the treatment of the SARS-CoV-2 and has the potential to become an essential medicine in the toolbox against the SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Animals , Antibody Affinity/immunology , Binding Sites , Epitopes/immunology , Humans , Neutralization Tests , Peptide Library , Protein Binding
17.
Biomolecules ; 10(12)2020 12 11.
Article in English | MEDLINE | ID: covidwho-1383863

ABSTRACT

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , COVID-19/virology , Click Chemistry , Humans , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/pharmacology
18.
Elife ; 102021 08 02.
Article in English | MEDLINE | ID: covidwho-1377103

ABSTRACT

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking viral receptor angiotensin-converting enzyme 2 (ACE2). The lead drug candidate possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD ~3000 times more tightly than ACE2 did and inhibited SARS-CoV-2 pseudovirus ~160 times more efficiently than ACE2 did. Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy against live SARS-CoV-2 infection in both hamster and mouse models. Unlike conventional antibodies, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented an excellent in vivo stability and a high tissue bioavailability. As effective and inexpensive drug candidates, Nanosota-1 may contribute to the battle against COVID-19.


Subject(s)
Antibodies, Viral/immunology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Single-Domain Antibodies/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Molecular , Pandemics , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/metabolism
19.
Front Immunol ; 12: 719037, 2021.
Article in English | MEDLINE | ID: covidwho-1354866

ABSTRACT

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.


Subject(s)
COVID-19 Serological Testing/methods , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/ultrastructure , Single-Domain Antibodies/immunology , Single-Domain Antibodies/ultrastructure , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Crystallography, X-Ray , Humans , Protein Domains , SARS-CoV-2/immunology
20.
Commun Biol ; 4(1): 366, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1351981

ABSTRACT

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.


Subject(s)
Chromatography, Gel , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Nanotechnology , Peptides/metabolism , Single-Domain Antibodies/immunology , Animals , Cryoelectron Microscopy , Cysteine Loop Ligand-Gated Ion Channel Receptors/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors/immunology , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Oryzias/genetics , Oryzias/metabolism , Peptides/genetics , Peptides/immunology , Protein Stability , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spectrometry, Fluorescence , Temperature , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism
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