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1.
Nat Commun ; 12(1): 3172, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1550281

ABSTRACT

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Subject(s)
Nerve Tissue Proteins/ultrastructure , Neuropilin-1/ultrastructure , Receptors, Cell Surface/ultrastructure , Semaphorin-3A/ultrastructure , Animals , COS Cells , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neuropilin-1/genetics , Neuropilin-1/isolation & purification , Neuropilin-1/metabolism , Protein Binding/genetics , Protein Domains/genetics , Protein Multimerization/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Semaphorin-3A/genetics , Semaphorin-3A/isolation & purification , Semaphorin-3A/metabolism
2.
Viruses ; 13(12)2021 11 29.
Article in English | MEDLINE | ID: covidwho-1542799

ABSTRACT

As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have a potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein, along with certain mutations in other regions within the S protein as specific variants, in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Diagnostic Tests, Routine/methods , SARS-CoV-2/genetics , Binding Sites/genetics , COVID-19/diagnosis , COVID-19/virology , Humans , Mutation , Protein Domains/genetics , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Sequence Analysis, DNA , Spike Glycoprotein, Coronavirus/genetics
3.
Front Immunol ; 12: 757691, 2021.
Article in English | MEDLINE | ID: covidwho-1463478

ABSTRACT

The increase in confirmed COVID-19 cases and SARS-CoV-2 variants calls for the development of safe and broad cross-protective vaccines. The RBD of the spike protein was considered to be a safe and effective candidate antigen. However, the low immunogenicity limited its application in vaccine development. Herein, we designed and obtained an RBD heptamer (mHla-RBD) based on a carrier protein-aided assembly strategy. The molecular weight of mHla-RBD is up to 450 kDa, approximately 10 times higher than that of the RBD monomer. When formulated with alum adjuvant, mHla-RBD immunization significantly increased the immunogenicity of RBD, as indicated by increased titers of RBD-specific antibodies, neutralizing antibodies, Th2 cellular immune response, and pseudovirus neutralization activity, when compared to RBD monomer. Furthermore, we confirmed that RBD-specific antibodies predominantly target conformational epitopes, which was approximately 200 times that targeting linear epitopes. Finally, a pseudovirus neutralization assay revealed that neutralizing antibodies induced by mHla-RBD against different SARS-CoV-2 variants were comparable to those against the wild-type virus and showed broad-spectrum neutralizing activity toward different SARS-CoV-2 variants. Our results demonstrated that mHla-RBD is a promising candidate antigen for development of SARS-CoV-2 vaccines and the mHla could serve as a universal carrier protein for antigen design.


Subject(s)
Bacterial Proteins/metabolism , COVID-19 Vaccines/immunology , COVID-19/immunology , Carrier Proteins/metabolism , Hemolysin Proteins/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Th2 Cells/immunology , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Broadly Neutralizing Antibodies/metabolism , Cell Line , Escherichia coli Proteins , Humans , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
4.
Viruses ; 13(10)2021 10 02.
Article in English | MEDLINE | ID: covidwho-1444335

ABSTRACT

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , Arabinonucleotides/pharmacology , SARS-CoV-2/drug effects , Virus Attachment/drug effects , COVID-19/drug therapy , Humans , Protein Binding/drug effects , Protein Domains/drug effects , Protein Domains/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
6.
Nat Commun ; 12(1): 5654, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440471

ABSTRACT

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites/genetics , COVID-19/mortality , COVID-19/virology , Disease Models, Animal , Female , Humans , Male , Mice , Protein Binding/genetics , Protein Domains/genetics , SARS-CoV-2/genetics , Severity of Illness Index , Spike Glycoprotein, Coronavirus/genetics
7.
J Biol Chem ; 297(4): 101208, 2021 10.
Article in English | MEDLINE | ID: covidwho-1415532

ABSTRACT

Emergence of new severe acute respiratory syndrome coronavirus 2 variants has raised concerns related to the effectiveness of vaccines and antibody therapeutics developed against the unmutated wildtype virus. Here, we examined the effect of the 12 most commonly occurring mutations in the receptor-binding domain of the spike protein on its expression, stability, activity, and antibody escape potential. Stability was measured using thermal denaturation, and the activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding to the human angiotensin-converting enzyme 2 and to neutralizing human antibody CC12.1, respectively. Our results show that mutants differ in their expression levels. Of the eight best-expressed mutants, two (N501Y and K417T/E484K/N501Y) showed stronger affinity to angiotensin-converting enzyme 2 compared with the wildtype, whereas four (Y453F, S477N, T478I, and S494P) had similar affinity and two (K417N and E484K) had weaker affinity than the wildtype. Compared with the wildtype, four mutants (K417N, Y453F, N501Y, and K417T/E484K/N501Y) had weaker affinity for the CC12.1 antibody, whereas two (S477N and S494P) had similar affinity, and two (T478I and E484K) had stronger affinity than the wildtype. Mutants also differ in their thermal stability, with the two least stable mutants showing reduced expression. Taken together, these results indicate that multiple factors contribute toward the natural selection of variants, and all these factors need to be considered to understand the evolution of the virus. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that variant.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Antigen-Antibody Reactions , COVID-19/pathology , COVID-19/virology , HEK293 Cells , Humans , Polymorphism, Single Nucleotide , Protein Binding , Protein Domains/genetics , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Transition Temperature
8.
Protein Expr Purif ; 179: 105802, 2021 03.
Article in English | MEDLINE | ID: covidwho-1386444

ABSTRACT

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection.


Subject(s)
COVID-19/blood , Protein Domains/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/isolation & purification , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/virology , Enzyme-Linked Immunosorbent Assay , Humans , Protein Binding/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/genetics
9.
Angew Chem Int Ed Engl ; 60(24): 13280-13286, 2021 06 07.
Article in English | MEDLINE | ID: covidwho-1384109

ABSTRACT

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5' cap is important for mRNA translation and immune response and can be naturally methylated at different positions by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N6 -position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo-enzymatic propargylation enabled production of site-specifically modified reporter-mRNAs. These cap-propargylated mRNAs were efficiently translated and showed ≈3-fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS-CoV-2-a currently tested epitope for mRNA vaccination-was used. Site-specific chemo-enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.


Subject(s)
RNA Caps/immunology , RNA, Messenger/immunology , COVID-19/pathology , COVID-19/virology , Chromatography, High Pressure Liquid , Genes, Reporter , HEK293 Cells , Humans , Mass Spectrometry , Methylation , Methyltransferases/metabolism , Protein Biosynthesis , Protein Domains/genetics , Protein Domains/immunology , RNA Caps/analysis , RNA Caps/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , S-Adenosylmethionine/chemistry , S-Adenosylmethionine/immunology , S-Adenosylmethionine/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Nucleic Acids Res ; 49(9): 5382-5392, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1387965

ABSTRACT

The emergence of SARS-CoV-2 infection has posed unprecedented threat to global public health. The virus-encoded non-structural protein 14 (nsp14) is a bi-functional enzyme consisting of an exoribonuclease (ExoN) domain and a methyltransferase (MTase) domain and plays a pivotal role in viral replication. Here, we report the structure of SARS-CoV-2 nsp14-ExoN domain bound to its co-factor nsp10 and show that, compared to the SARS-CoV nsp10/nsp14-full-length complex, SARS-CoV-2 nsp14-ExoN retains an integral exoribonuclease fold and preserves an active configuration in the catalytic center. Analysis of the nsp10/nsp14-ExoN interface reveals a footprint in nsp10 extensively overlapping with that observed in the nsp10/nsp16 structure. A marked difference in the co-factor when engaging nsp14 and nsp16 lies in helix-α1', which is further experimentally ascertained to be involved in nsp14-binding but not in nsp16-engagement. Finally, we also show that nsp10/nsp14-ExoN is enzymatically active despite the absence of nsp14-MTase domain. These data demonstrate that SARS-CoV-2 nsp10/nsp14-ExoN functions as an exoribonuclease with both structural and functional integrity.


Subject(s)
Biocatalysis , Exoribonucleases/chemistry , Exoribonucleases/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Exoribonucleases/genetics , Guanine , Methyltransferases/chemistry , Methyltransferases/deficiency , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Protein Domains/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
11.
J Proteome Res ; 19(12): 4844-4856, 2020 12 04.
Article in English | MEDLINE | ID: covidwho-1387125

ABSTRACT

Despite considerable research progress on SARS-CoV-2, the direct zoonotic origin (intermediate host) of the virus remains ambiguous. The most definitive approach to identify the intermediate host would be the detection of SARS-CoV-2-like coronaviruses in wild animals. However, due to the high number of animal species, it is not feasible to screen all the species in the laboratory. Given that binding to ACE2 proteins is the first step for the coronaviruses to invade host cells, we propose a computational pipeline to identify potential intermediate hosts of SARS-CoV-2 by modeling the binding affinity between the Spike receptor-binding domain (RBD) and host ACE2. Using this pipeline, we systematically examined 285 ACE2 variants from mammals, birds, fish, reptiles, and amphibians, and found that the binding energies calculated for the modeled Spike-RBD/ACE2 complex structures correlated closely with the effectiveness of animal infection as determined by multiple experimental data sets. Built on the optimized binding affinity cutoff, we suggest a set of 96 mammals, including 48 experimentally investigated ones, which are permissive to SARS-CoV-2, with candidates from primates, rodents, and carnivores at the highest risk of infection. Overall, this work not only suggests a limited range of potential intermediate SARS-CoV-2 hosts for further experimental investigation, but also, more importantly, it proposes a new structure-based approach to general zoonotic origin and susceptibility analyses that are critical for human infectious disease control and wildlife protection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Binding Sites/genetics , COVID-19/pathology , COVID-19/virology , Host-Pathogen Interactions/genetics , Humans , Mammals/genetics , Mammals/virology , Pandemics , Protein Binding/genetics , Protein Domains/genetics , SARS-CoV-2/pathogenicity , Viral Zoonoses/genetics , Viral Zoonoses/virology
12.
Int J Mol Sci ; 22(17)2021 Aug 26.
Article in English | MEDLINE | ID: covidwho-1374427

ABSTRACT

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.


Subject(s)
COVID-19/transmission , Coronavirus Nucleocapsid Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , SARS-CoV-2/pathogenicity , Animals , Binding Sites/genetics , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Humans , Interferon Type I/immunology , Interferon Type I/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/isolation & purification , Mutation , Phosphoproteins/genetics , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , Phosphoproteins/metabolism , Phosphorylation , Protein Binding/genetics , Protein Binding/immunology , Protein Domains/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells
13.
Front Immunol ; 12: 710263, 2021.
Article in English | MEDLINE | ID: covidwho-1315952

ABSTRACT

The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufacturing. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as second-generation vaccine candidates, as they feature robust manufacturing characteristics and have shown promising immunogenicity in preclinical models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid binding pocket. Screening of several designs led to the selection of two lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°C, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clinical trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunization Schedule , Immunogenicity, Vaccine , Mutation , Protein Domains/genetics , Protein Domains/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/virology , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Linoleic Acids , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Treatment Outcome , Vero Cells
14.
Cell Rep ; 36(4): 109433, 2021 07 27.
Article in English | MEDLINE | ID: covidwho-1300649

ABSTRACT

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , COVID-19/virology , Humans , Mutation/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/immunology
15.
J Phys Chem Lett ; 12(25): 5987-5993, 2021 Jul 01.
Article in English | MEDLINE | ID: covidwho-1279809

ABSTRACT

The rapid and relentless emergence of novel highly transmissible SARS-CoV-2 variants, possibly decreasing vaccine efficacy, currently represents a formidable medical and societal challenge. These variants frequently hold mutations on the Spike protein's receptor-binding domain (RBD), which, binding to the angiotensin-converting enzyme 2 (ACE2) receptor, mediates viral entry into host cells. Here, all-atom molecular dynamics simulations and dynamical network theory of the wild-type and mutant RBD/ACE2 adducts disclose that while the N501Y mutation (UK variant) enhances the Spike's binding affinity toward ACE2, the concomitant N501Y, E484K, and K417N mutations (South African variant) aptly adapt to increase SARS-CoV-2 propagation via a two-pronged strategy: (i) effectively grasping ACE2 through an allosteric signaling between pivotal RBD structural elements and (ii) impairing the binding of antibodies elicited by infected or vaccinated patients. This information unlocks the molecular terms and evolutionary strategies underlying the increased virulence of emerging SARS-CoV-2 variants, setting the basis for developing the next-generation anti-COVID-19 therapeutics.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Humans , Molecular Dynamics Simulation , Mutation , Protein Binding/genetics , Protein Domains/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics
16.
Sci Rep ; 11(1): 12740, 2021 06 17.
Article in English | MEDLINE | ID: covidwho-1275953

ABSTRACT

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.


Subject(s)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19/metabolism , Drug Discovery/methods , Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Signal Transduction/drug effects , Amino Acid Sequence , COVID-19/virology , Humans , Mutation , Protein Binding/drug effects , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
17.
Phys Chem Chem Phys ; 23(24): 13752-13759, 2021 Jun 28.
Article in English | MEDLINE | ID: covidwho-1270680

ABSTRACT

SARS-CoV-2 has recently caused an epidemic in humans and poses a huge threat to global public health. As a primary receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2) exists in different hosts that are in close contact with humans, especially cats and dogs. However, the underlying mechanism of how the spike receptor binding domain (RBD) of SARS-CoV-2 cooperates with human ACE2 (hACE2), cat ACE2 (cACE2) and dog ACE2 (dACE2) and the variation in binding remains largely unsolved. Therefore, we explored the binding behavior of the spike RBD with cACE2, dACE2 and hACE2 via all-atom molecular dynamics simulations. In accordance with the binding free energies and residue interactions, the spike RBD has respective binding specificities with cACE2, dACE2 and hACE2, and the binding affinities decrease in the order of hACE2, cACE2, dACE2, mainly due to changes in the amino acids Q24L, H34Y, and M82T in cACE2 or dACE2. Furthermore, alanine scanning analysis results validated some key residues of the spike RBD interact with ACE2 and provided clues to the variation of amino acid that could influence the transmissibility or immune responses of SARS-CoV-2. Decreasing dynamic correlations strengths of ACE2 with the RBD were found in all hACE2-RBD, cACE2-RBD and dACE2-RBD systems. The ACE2 protein shows variable motion modes across the zinc metallopeptidase domain, which induces different interactions between ACE2 and the RBD. Our studies reveal that the motion pattern of the zinc metallopeptidase domain is critical to the binding behavior of RBD with ACE2. These findings could aid our understanding of selective recognition involving various ACE2 with the SARS-CoV-2 spike and shed further light on the binding mechanisms.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , Cats , Dogs , Humans , Molecular Dynamics Simulation , Mutation , Principal Component Analysis , Protein Binding/genetics , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics
18.
Molecules ; 26(11)2021 May 27.
Article in English | MEDLINE | ID: covidwho-1256613

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Discovery , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/therapeutic use , Aurintricarboxylic Acid/pharmacology , Aurintricarboxylic Acid/therapeutic use , COVID-19/virology , Chlorocebus aethiops , Ellagic Acid/pharmacology , Ellagic Acid/therapeutic use , Heparin/pharmacology , Heparin/therapeutic use , High-Throughput Screening Assays , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Protein Domains/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
19.
Sci Rep ; 11(1): 11046, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1246388

ABSTRACT

Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/therapy , Immunoglobulin G/metabolism , Immunotherapy/methods , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/immunology , Cells, Cultured , Epitopes , Humans , Immunoglobulin G/immunology , Pandemics , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
20.
Nat Commun ; 12(1): 3109, 2021 05 25.
Article in English | MEDLINE | ID: covidwho-1243298

ABSTRACT

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody Formation , COVID-19/blood , Female , Gene Expression , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Mutation , Neutralization Tests , Protein Binding , Protein Domains/genetics , Receptors, Coronavirus/metabolism , Recombinant Proteins , SARS-CoV-2/genetics , Saliva/immunology , Saliva/virology
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