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1.
Methods Mol Biol ; 2099: 21-37, 2020.
Article in English | MEDLINE | ID: covidwho-1292545

ABSTRACT

The coronavirus spike envelope glycoprotein is an essential viral component that mediates virus entry events. Biochemical assessment of the spike protein is critical for understanding structure-function relationships and the roles of the protein in the viral life cycle. Coronavirus spike proteins are typically proteolytically processed and activated by host cell enzymes such as trypsin-like proteases, cathepsins, or proprotein-convertases. Analysis of coronavirus spike proteins by western blot allows the visualization and assessment of proteolytic processing by endogenous or exogenous proteases. Here, we present a method based on western blot analysis to investigate spike protein proteolytic cleavage by transient transfection of HEK-293 T cells allowing expression of the spike protein of the highly pathogenic Middle East respiratory syndrome coronavirus in the presence or absence of a cellular trypsin-like transmembrane serine protease, matriptase. Such analysis enables the characterization of cleavage patterns produced by a host protease on a coronavirus spike glycoprotein.


Subject(s)
Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Blotting, Western , Cell Line , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Protein Processing, Post-Translational , Proteolysis , Serine Endopeptidases/metabolism , Virus Internalization
2.
ACS Nano ; 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1269369

ABSTRACT

With an incubation time of about 5 days, early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to control the spread of the coronavirus disease 2019 (COVID-19) that killed more than 3 million people in its first 1.5 years. Here, we report on the modification of the dopant density and the phononic energy of antibody-coupled graphene when it interfaces with SARS-CoV-2 spike protein. This graphene chemeo-phononic system was able to detect SARS-CoV-2 spike protein at the limit of detection of ∼3.75 and ∼1 fg/mL in artificial saliva and phosphate-buffered saline, respectively. It also exhibited selectivity over proteins in saliva and MERS-CoV spike protein. Since the change in graphene phononics is monitored instead of the phononic signature of the analyte, this optical platform can be replicated for other COVID variants and specific-binding-based biodetection applications.

3.
Nat Biomed Eng ; 5(7): 666-677, 2021 07.
Article in English | MEDLINE | ID: covidwho-1241951

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody-SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 µl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads.


Subject(s)
Biosensing Techniques/methods , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Nanotechnology/methods , SARS Virus/pathogenicity , COVID-19/virology , Humans , Single-Domain Antibodies/immunology
4.
Arch Immunol Ther Exp (Warsz) ; 69(1): 5, 2021 Mar 06.
Article in English | MEDLINE | ID: covidwho-1118194

ABSTRACT

Coronaviruses share conservative spike protein (S) on their enveloped membrane surface, where S1 subunit recognizes and binds the cellular receptor, and the S2 subunit mediates membrane fusion. This similarity raises the question: does coronaviral infection by one create protection to others? Convalescent SARS-CoV-2 (COVID-19) sera were tested for cross reactivity with peptides from Middle East respiratory syndrome coronavirus (MERS-CoV) which shares 74% homology. Our results showed significant cross-reactivity with a peptide of the heptad repeat 2 (HR2) domain of the MERS-CoV spike protein. Sera samples of 47 validated seropositive convalescent COVID-19 patients and 40 sera samples of control patients, collected in pre-COVID time were used to establish cross-bind reactivity with the MERS-CoV peptide. Significantly stronger binding (p < 0.0001) was observed for IgG antibodies in convalescent COVID-19 patients compared to the control group. In ELISA, MERS-CoV peptide helps to discriminate post-COVID-19 populations and non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection can recognize and probably decrease severity of MERS-CoV and other coronaviral infections. The high homology of the spike protein domain also suggests that the opposite effect can be true: coronaviral infections produce cross-reactive antibodies effective against SARS-CoV-2. The collected data prove that despite the core HR2 region is hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections and developing vaccines effective even after possible viral mutations.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Convalescence , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Cross Reactions , Humans , Middle East Respiratory Syndrome Coronavirus/immunology , SARS Virus/immunology
5.
mBio ; 12(2)2021 03 02.
Article in English | MEDLINE | ID: covidwho-1115091

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.


Subject(s)
Proline/chemistry , SARS-CoV-2/immunology , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , COVID-19 Vaccines/therapeutic use , Mice , Mutation , Spike Glycoprotein, Coronavirus/chemistry
6.
Int J Mol Sci ; 22(4)2021 Feb 15.
Article in English | MEDLINE | ID: covidwho-1085072

ABSTRACT

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19-663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs' activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08-1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , Immunoglobulin Fab Fragments/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Binding Sites , Humans , Immunoglobulin G/immunology , Peptide Library , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry
7.
J King Saud Univ Sci ; 33(2): 101335, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1023655

ABSTRACT

Coronaviruses M proteins are well-represented in the major protein component of the viral envelope. During the viral assembly, they play an important role by association with all other viral structural proteins. Despite their crucial functions, very little information regarding the structures and functions of M proteins is available. Here we utilize bioinformatic tools from available sequences and 3D structures of SARS-CoV, SARS-CoV2, and MERS-CoV M proteins in order to predict potential B-cell epitopes and assessing antibody binding affinity. Such study aims to aid finding more effective vaccines and recognize neutralizing antibodies. we found some rather exciting differences between SARS-COV-2, SARS-Cov and MERS-CoV M proteins. Two SARS-CoV-2 peptides with significant antigen presentation scores for human cell surface proteins have been identified. The results reveal that N-terminal domains of M proteins of SARS-CoV and SARS-CoV2 are translocated (outside) whereas it is inside (cytoplasmic side) in MERS-CoV.

8.
Molecules ; 25(22)2020 Nov 18.
Article in English | MEDLINE | ID: covidwho-934509

ABSTRACT

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.


Subject(s)
Angiotensin-Converting Enzyme 2/isolation & purification , Dipeptidyl Peptidase 4/isolation & purification , Spike Glycoprotein, Coronavirus/isolation & purification , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Cloning, Molecular , Dipeptidyl Peptidase 4/biosynthesis , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Humans , Kinetics , Models, Molecular , Plasmids/chemistry , Plasmids/metabolism , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sf9 Cells , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spodoptera , Surface Plasmon Resonance
9.
Sci Rep ; 10(1): 20085, 2020 11 18.
Article in English | MEDLINE | ID: covidwho-933722

ABSTRACT

The COVID-19 pandemic is a worldwide health emergency which calls for an unprecedented race for vaccines and treatment. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein, S-2P. To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of neutralizing antibodies in sera of immunized mice against pseudotyped lentivirus reporter or live wild-type SARS-CoV-2. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018 and alum as a candidate vaccine to prevent COVID-19 disease.


Subject(s)
COVID-19 Vaccines/immunology , Immunogenicity, Vaccine , Spike Glycoprotein, Coronavirus/immunology , Adjuvants, Immunologic/therapeutic use , Aluminum Hydroxide/therapeutic use , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , CHO Cells , COVID-19 Vaccines/adverse effects , COVID-19 Vaccines/therapeutic use , Cricetinae , Cricetulus , Cytokines/blood , Cytokines/metabolism , Female , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Oligodeoxyribonucleotides/therapeutic use , Rats , Rats, Sprague-Dawley , Spleen/immunology , Th1 Cells/immunology
10.
Vaccines (Basel) ; 8(4)2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-902683

ABSTRACT

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 and causes severe and often fatal acute respiratory illness in humans. No approved prophylactic and therapeutic interventions are currently available. In this study, we have developed egg yolk antibodies (immunoglobulin Y (IgY)) specific for MERS-CoV spike protein (S1) in order to evaluate their neutralizing efficiency against MERS-CoV infection. S1-specific immunoglobulins were produced by injecting chickens with purified recombinant S1 protein of MERS-CoV at a high titer (5.7 mg/mL egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated that the IgY antibody specifically bound to the MERS-CoV S1 protein. Anti-S1 antibodies were also able to recognize MERS-COV inside cells, as demonstrated by an immunofluorescence assay. Plaque reduction and microneutralization assays showed the neutralization of MERS-COV in Vero cells by anti-S1 IgY antibodies and non-significantly reduced virus titers in the lungs of MERS-CoV-infected mice during early infection, with a nonsignificant decrease in weight loss. However, a statistically significant (p = 0.0196) quantitative reduction in viral antigen expression and marked reduction in inflammation were observed in lung tissue. Collectively, our data suggest that the anti-MERS-CoV S1 IgY could serve as a potential candidate for the passive treatment of MERS-CoV infection.

11.
Sci Rep ; 10(1): 16944, 2020 10 09.
Article in English | MEDLINE | ID: covidwho-842355

ABSTRACT

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin's ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.


Subject(s)
Betacoronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Furin/metabolism , Phosphorylation , Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
12.
J Virol ; 94(14)2020 07 01.
Article in English | MEDLINE | ID: covidwho-840682

ABSTRACT

Autonomously replicating subgenomic Bungowannah virus (BuPV) RNAs (BuPV replicons) with deletions of the genome regions encoding the structural proteins C, ERNS, E1, and E2 were constructed on the basis of an infectious cDNA clone of BuPV. Nanoluciferase (Nluc) insertion was used to compare the replication efficiencies of all constructs after electroporation of in vitro-transcribed RNA from the different clones. Deletion of C, E1, E2, or the complete structural protein genome region (C-ERNS-E1-E2) prevented the production of infectious progeny virus, whereas deletion of ERNS still allowed the generation of infectious particles. However, those ΔERNS viral particles were defective in virus assembly and/or egress and could not be further propagated for more than three additional passages in porcine SK-6 cells. These "defective-in-third-cycle" BuPV ΔERNS mutants were subsequently used to express the classical swine fever virus envelope protein E2, the N-terminal domain of the Schmallenberg virus Gc protein, and the receptor binding domain of the Middle East respiratory syndrome coronavirus spike protein. The constructs could be efficiently complemented and further passaged in SK-6 cells constitutively expressing the BuPV ERNS protein. Importantly, BuPVs are able to infect a wide variety of target cell lines, allowing expression in a very wide host spectrum. Therefore, we suggest that packaged BuPV ΔERNS replicon particles have potential as broad-spectrum viral vectors.IMPORTANCE The proteins NPRO and ERNS are unique for the genus Pestivirus, but only NPRO has been demonstrated to be nonessential for in vitro growth. While this was also speculated for ERNS, it has always been previously shown that pestivirus replicons with deletions of the structural proteins ERNS, E1, or E2 did not produce any infectious progeny virus in susceptible host cells. Here, we demonstrated for the first time that BuPV ERNS is dispensable for the generation of infectious virus particles but still important for efficient passaging. The ERNS-defective BuPV particles showed clearly limited growth in cell culture but were capable of several rounds of infection, expression of foreign genes, and highly efficient trans-complementation to rescue virus replicon particles (VRPs). The noncytopathic characteristics and the absence of preexisting immunity to BuPV in human populations and livestock also provide a significant benefit for a possible use, e.g., as a vector vaccine platform.


Subject(s)
Pestivirus Infections/virology , Pestivirus/physiology , RNA, Viral , Viral Envelope Proteins/metabolism , Virus Replication , Gene Deletion , Gene Expression , Genes, Reporter , Genetic Engineering , Host-Pathogen Interactions , Pestivirus Infections/immunology , Replicon , Viral Envelope Proteins/genetics , Virion , Virus Assembly
13.
Sci Rep ; 10(1): 16615, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-834915

ABSTRACT

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Subject(s)
Antibodies, Viral/analysis , Antibody-Dependent Cell Cytotoxicity/immunology , Coronavirus Infections/immunology , Cytotoxicity Tests, Immunologic/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/virology , Genes, Reporter , HEK293 Cells , Humans , Jurkat Cells , Luciferases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , NFATC Transcription Factors/genetics , Receptors, IgG/genetics , Receptors, IgG/immunology , Response Elements , Spike Glycoprotein, Coronavirus/metabolism , Transfection
14.
Microb Risk Anal ; 16: 100140, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-779468

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) infect the human respiratory tract. A prototype thermodynamic equilibrium model is presented here for the probability of the virions getting through the mucus barrier and infecting epithelial cells based on the binding affinity (Kmucin) of the virions to mucin molecules in the mucus and parameters for binding and infection of the epithelial cell. Both MERS-CoV and SARS-CoV-2 bind strongly to their cellular receptors, DDP4 and ACE2, respectively, and infect very efficiently both bronchus and lung ex vivo cell cultures which are not protected by a mucus barrier. According to the model, mucin binding could reduce the infectivity for MERS-CoV compared to SARS-CoV-2 by at least 100-fold depending on the magnitude of Kmucin. Specifically Kmucin values up to 106 M-1 have little protective effect and thus the mucus barrier would not remove SARS-CoV-2 which does not bind to sialic acids (SA) and hence would have a very low Kmucin. Depending on the viability of individual virions, the ID50 for SARS-CoV-2 is estimated to be ~500 virions (viral RNA genomic copies) representing 1 to 2 pfu. In contrast MERS-CoV binds both SA and human mucin and a Kmucin of 5 × 109 M-1 as reported for lectins would mop up 99.83% of the virus according to the model with the ID50 for MERS-CoV estimated to be ~295,000 virions (viral RNA genomic copies) representing 819 pfu. This could in part explain why MERS-CoV is poorly transmitted from human to human compared to SARS-CoV-2. Some coronaviruses use an esterase to escape the mucin, although MERS-CoV does not. Instead, it is shown here that "clustering" of virions into single aerosol particles as recently reported for rotavirus in extracellular vesicles could provide a co-operative mechanism whereby MERS-CoV could theoretically overcome the mucin barrier locally and a small proportion of 10 µm diameter aerosol particles could contain ~70 virions based on reported maximum levels in saliva. Although recent evidence suggests SARS-CoV-2 initiates infection in the nasal epithelium, the thermodynamic equilibrium models presented here could complement published approaches for modelling the physical entry of pathogens to the lung based on the fate and transport of the pathogen particles (as for anthrax spores) to develop a dose-response model for aerosol exposure to respiratory viruses. This would enable the infectivity through aerosols to be defined based on molecular parameters as well as physical parameters. The role of the spike proteins of MERS-CoV and SARS-CoV-2 binding to SA and heparan sulphate, respectively, may be to aid non-specific attachment to the host cell. It is proposed that a high Kmucin is the cost for subsequent binding of MERS-CoV to SAs on the cell surface to partially overcome the unfavourable entropy of immobilisation as the virus adopts the correct orientation for spike protein interactions with its protein cellular receptor DPP4.

15.
Viruses ; 12(9)2020 08 19.
Article in English | MEDLINE | ID: covidwho-721525

ABSTRACT

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.


Subject(s)
Betacoronavirus/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Sialic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Amino Sugars/metabolism , Betacoronavirus/physiology , Binding Sites , Models, Molecular , Molecular Docking Simulation , Molecular Dynamics Simulation , N-Acetylneuraminic Acid/metabolism , Protein Binding , Protein Domains , Receptors, Coronavirus , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS Virus/chemistry , SARS-CoV-2 , Sialyl Lewis X Antigen/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Virus Internalization
16.
Viruses ; 12(6)2020 06 03.
Article in English | MEDLINE | ID: covidwho-532726

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.


Subject(s)
Camelus/virology , Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Betacoronavirus/classification , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Biological Evolution , DNA, Complementary/chemistry , DNA, Viral/chemistry , Epitopes/analysis , Epitopes/chemistry , Epitopes/genetics , Gene Library , Humans , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Phylogeny , RNA, Viral/analysis , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Saudi Arabia
17.
PLoS One ; 15(5): e0232757, 2020.
Article in English | MEDLINE | ID: covidwho-209798

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection and continues to infect humans, thereby contributing to a high mortality rate (34.3% in 2019). In the absence of an available licensed vaccine and antiviral agent, therapeutic human antibodies have been suggested as candidates for treatment. In this study, human monoclonal antibodies were isolated by sorting B cells from patient's PBMC cells with prefusion stabilized spike (S) probes and a direct immunoglobulin cloning strategy. We identified six receptor-binding domain (RBD)-specific and five S1 (non-RBD)-specific antibodies, among which, only the RBD-specific antibodies showed high neutralizing potency (IC50 0.006-1.787 µg/ml) as well as high affinity to RBD. Notably, passive immunization using a highly potent antibody (KNIH90-F1) at a relatively low dose (2 mg/kg) completely protected transgenic mice expressing human DPP4 against MERS-CoV lethal challenge. These results suggested that human monoclonal antibodies isolated by using the rationally designed prefusion MERS-CoV S probe could be considered potential candidates for the development of therapeutic and/or prophylactic antiviral agents for MERS-CoV human infection.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Viral/pharmacology , Coronavirus Infections/drug therapy , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Dipeptidyl Peptidase 4/genetics , Humans , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Republic of Korea , Vero Cells
18.
mBio ; 11(2)2020 04 07.
Article in English | MEDLINE | ID: covidwho-38355

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 104 PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERSMA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 106 PFU UV-inactivated MERSMA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.


Subject(s)
Coronavirus Infections/prevention & control , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Administration, Intranasal , Animals , Antibodies, Viral/blood , Coronavirus Infections/immunology , Coronavirus Infections/mortality , Disease Models, Animal , Immunization , Mice , Mice, Inbred C57BL , Parainfluenza Virus 5/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
19.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(6): 614-619, 2020 Jun 06.
Article in Chinese | MEDLINE | ID: covidwho-27060

ABSTRACT

The outbreak of 2019-novel coronavirus (2019-nCoV) infection poses a serious threat to global public health. Vaccination is an effective way to prevent the epidemic of the virus. 2019-nCoV along with severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) belong to the same ß-genus of coronavirus family. Basing on the previous experience and the technical platform of developing SARS-CoV and MERS-CoV vaccines, scientists from all over the world are working hard and quickly on the related fields. There are substantial progress in these fields including characterizing the 2019-nCoV virus, identification of candidate antigens and epitopes, establishment of animal models, characterizing the immune responses, and the design of vaccines. The development of 2019-nCoV vaccines covers all types: inactivated virus vaccine, recombinant protein vaccine, viral vector-based vaccine, mRNA vaccine, and DNA vaccine, et al. As of March 2020, two 2019-nCoV vaccines have entered phase I clinical trials. One is named as Ad5-nCoV developed by the Chinese Institute of Biotechnology of the Academy of Military Medical Sciences and Tianjin Cansino Biotechnology Inc. Ad5-nCoV is based on the replication-defective adenovirus type 5 as the vector to express 2019-nCoV spike protein. The another vaccine is mRNA-1273 developed by the National Institute of Allergy and Infectious Diseases and Moderna, Inc.. RNA-1273 is an mRNA vaccine expressing 2019-nCoV spike protein. Although the rapid development of 2019-nCoV vaccine, it still faces many unknown challenges, including the antigenic characteristics of the 2019-nCoV, the influence of antigenic variation, the protective immune response of host, the protection of the elderly population, and the downstream manufacturing process of the new vaccine. The safety and efficacy of vaccines are the first priority for vaccine development and should be carefully evaluated.


Subject(s)
Biomedical Research/organization & administration , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/epidemiology , Humans , Pneumonia, Viral/epidemiology
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