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1.
Biophys J ; 120(14): 2793-2804, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1607417

ABSTRACT

The ongoing coronavirus disease 19 (COVID-19) pandemic has infected millions of people, claimed hundreds of thousands of lives, and made a worldwide health emergency. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanism of infection is crucial in the development of potential therapeutics and vaccines. The infection process is triggered by direct binding of the SARS-CoV-2 receptor-binding domain (RBD) to the host-cell receptor angiotensin-converting enzyme 2 (ACE2). Many efforts have been made to design or repurpose therapeutics to deactivate the RBD or ACE2 and prevent the initial binding. In addition to direct inhibition strategies, small chemical compounds might be able to interfere and destabilize the metastable, prefusion complex of ACE2-RBD. This approach can be employed to prevent the further progress of virus infection at its early stages. In this study, molecular docking was employed to analyze the binding of two chemical compounds, SSAA09E2 and Nilotinib, with the druggable pocket of the ACE2-RBD complex. The structural changes as a result of the interference with the ACE2-RBD complex were analyzed by molecular dynamics simulations. Results show that both Nilotinib and SSAA09E2 can induce significant conformational changes in the ACE2-RBD complex, intervene with the hydrogen bonds, and influence the flexibility of proteins. Moreover, essential dynamics analysis suggests that the presence of small molecules can trigger large-scale conformational changes that may destabilize the ACE2-RBD complex.


Subject(s)
COVID-19 , Molecular Dynamics Simulation , Angiotensin-Converting Enzyme 2 , Humans , Molecular Docking Simulation , Peptidyl-Dipeptidase A , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
2.
Biophys J ; 120(14): 2785-2792, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1603521

ABSTRACT

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.


Subject(s)
COVID-19 , Peptidyl-Dipeptidase A , Angiotensin-Converting Enzyme 2 , Humans , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
3.
Int J Mol Med ; 49(2)2022 02.
Article in English | MEDLINE | ID: covidwho-1594678

ABSTRACT

The pathophysiology of coronavirus disease 2019 (COVID­19) is mainly dependent on the underlying mechanisms that mediate the entry of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) into the host cells of the various human tissues/organs. Recent studies have indicated a higher order of complexity of the mechanisms of infectivity, given that there is a wide­repertoire of possible cell entry mediators that appear to co­localise in a cell­ and tissue­specific manner. The present study provides an overview of the 'canonical' SARS­CoV­2 mediators, namely angiotensin converting enzyme 2, transmembrane protease serine 2 and 4, and neuropilin­1, expanding on the involvement of novel candidates, including glucose­regulated protein 78, basigin, kidney injury molecule­1, metabotropic glutamate receptor subtype 2, ADAM metallopeptidase domain 17 (also termed tumour necrosis factor­α convertase) and Toll­like receptor 4. Furthermore, emerging data indicate that changes in microRNA (miRNA/miR) expression levels in patients with COVID­19 are suggestive of further complexity in the regulation of these viral mediators. An in silico analysis revealed 160 candidate miRNAs with potential strong binding capacity in the aforementioned genes. Future studies should concentrate on elucidating the association between the cellular tropism of the SARS­CoV­2 cell entry mediators and the mechanisms through which they might affect the clinical outcome. Finally, the clinical utility as a biomarker or therapeutic target of miRNAs in the context of COVID­19 warrants further investigation.


Subject(s)
COVID-19/metabolism , MicroRNAs/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , /metabolism , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , Neuropilin-1/genetics , Neuropilin-1/metabolism , Receptors, Virus/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Viral Tropism
4.
Biochemistry (Mosc) ; 86(7): 800-817, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1594970

ABSTRACT

COVID-19, a new human respiratory disease that has killed nearly 3 million people in a year since the start of the pandemic, is a global public health challenge. Its infectious agent, SARS-CoV-2, differs from other coronaviruses in a number of structural features that make this virus more pathogenic and transmissible. In this review, we discuss some important characteristics of the main SARS-CoV-2 surface antigen, the spike (S) protein, such as (i) ability of the receptor-binding domain (RBD) to switch between the "standing-up" position (open pre-fusion conformation) for receptor binding and the "lying-down" position (closed pre-fusion conformation) for immune system evasion; (ii) advantage of a high binding affinity of the RBD open conformation to the human angiotensin-converting enzyme 2 (ACE2) receptor for efficient cell entry; and (iii) S protein preliminary activation by the intracellular furin-like proteases for facilitation of the virus spreading across different cell types. We describe interactions between the S protein and cellular receptors, co-receptors, and antagonists, as well as a hypothetical mechanism of the homotrimeric spike structure destabilization that triggers the fusion of the viral envelope with the cell membrane at physiological pH and mediates the viral nucleocapsid entry into the cytoplasm. The transition of the S protein pre-fusion conformation to the post-fusion one on the surface of virions after their treatment with some reagents, such as ß-propiolactone, is essential, especially in relation to the vaccine production. We also compare the COVID-19 pathogenesis with that of severe outbreaks of "avian" influenza caused by the A/H5 and A/H7 highly pathogenic viruses and discuss the structural similarities between the SARS-CoV-2 S protein and hemagglutinins of those highly pathogenic strains. Finally, we touch on the prospective and currently used COVID-19 antiviral and anti-pathogenetic therapeutics, as well as recently approved conventional and innovative COVID-19 vaccines and their molecular and immunological features.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/genetics , COVID-19/metabolism , Humans , Influenza A virus/chemistry , Influenza A virus/genetics , Influenza A virus/metabolism , Influenza, Human/epidemiology , Influenza, Human/genetics , Influenza, Human/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
6.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1599475

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell-cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2' fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2' cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2' cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Membrane Fusion , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Mice , Protein Binding , Proteolysis , Virus Internalization
7.
J Am Soc Mass Spectrom ; 33(1): 181-188, 2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1596214

ABSTRACT

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for 5-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Discovery/methods , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Chalcones/chemistry , Chalcones/pharmacology , Drug Evaluation, Preclinical/methods , Fabaceae/chemistry , Humans , Ligands , Mass Spectrometry/methods , Molecular Docking Simulation , Protein Binding/drug effects , SARS-CoV-2/metabolism
8.
J Virol ; 94(13)2020 06 16.
Article in English | MEDLINE | ID: covidwho-1583223

ABSTRACT

Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.


Subject(s)
Calcium/metabolism , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Host-Pathogen Interactions , Ions/metabolism , Membrane Fusion , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Chlorocebus aethiops , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Models, Molecular , Mutation , Protein Binding , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship , Vero Cells , Virulence , Virus Assembly
9.
Signal Transduct Target Ther ; 6(1): 428, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1585884

ABSTRACT

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.


Subject(s)
COVID-19/metabolism , Cell Degranulation , Lung Injury/metabolism , Mast Cells/metabolism , Pulmonary Alveoli/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/genetics , Cell Line, Tumor , Female , Humans , Lung Injury/genetics , Lung Injury/virology , Macaca mulatta , Male , Mice, Inbred BALB C , Mice, Transgenic , Pulmonary Alveoli/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
10.
Sci Rep ; 11(1): 24336, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1585788

ABSTRACT

ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Bile Ducts/metabolism , Bile Ducts/virology , Binding Sites , COVID-19/pathology , COVID-19/virology , Humans , Lung/metabolism , Lung/virology , Microscopy, Fluorescence, Multiphoton , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization
11.
Commun Biol ; 4(1): 1389, 2021 12 16.
Article in English | MEDLINE | ID: covidwho-1585764

ABSTRACT

In light of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants potentially undermining humoral immunity, it is important to understand the fine specificity of the antiviral antibodies. We screened 20 COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. Importantly, mutations which typically occur in the B.1.351 "South African" variant, significantly reduced the binding of anti-RBD antibodies. Nine of 20 patients were critically ill and were considered high-risk (HR). These patients showed significantly higher levels of transforming growth factor beta (TGF-ß) and myeloid-derived suppressor cells (MDSC), and lower levels of CD4+ T cells expressing LAG-3 compared to standard-risk (SR) patients. HR patients evidenced significantly higher anti-S1/RBD IgG antibody levels and an increased neutralizing activity. Importantly, a large proportion of S protein-specific antibodies were glycosylation-dependent and we identified a number of immunodominant linear epitopes within the S1 and N proteins. Findings derived from this study will not only help us to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response but will also enable us to design more meaningful immunomonitoring methods for anti-COVID-19 vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Viral Proteins/immunology , Adaptive Immunity/immunology , Adult , Aged , COVID-19/virology , COVID-19 Vaccines/immunology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Female , Humans , Immunity, Humoral/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Male , Middle Aged , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
12.
MAbs ; 14(1): 2005507, 2022.
Article in English | MEDLINE | ID: covidwho-1585297

ABSTRACT

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


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Antibody Affinity/immunology , Antibody Specificity/immunology , CHO Cells , COVID-19/prevention & control , COVID-19/virology , Chromatography, High Pressure Liquid/methods , Circular Dichroism , Clone Cells , Cricetinae , Cricetulus , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/therapeutic use , Isoelectric Point , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism
13.
Mol Med ; 27(1): 162, 2021 12 27.
Article in English | MEDLINE | ID: covidwho-1582120

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel type b coronavirus responsible for the COVID-19 pandemic. With over 224 million confirmed infections with this virus and more than 4.6 million people dead because of it, it is critically important to define the immunological processes occurring in the human response to this virus and pathogenetic mechanisms of its deadly manifestation. This perspective focuses on the contribution of the recently discovered interaction of SARS-CoV-2 Spike protein with neuropilin 1 (NRP1) receptor, NRP1 as a virus entry receptor for SARS-CoV-2, its role in different physiologic and pathologic conditions, and the potential to target the Spike-NRP1 interaction to combat virus infectivity and severe disease manifestations.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Neuropilin-1/chemistry , Neuropilin-1/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , COVID-19/etiology , Comorbidity , Diabetes Mellitus/epidemiology , Diabetes Mellitus/virology , Female , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Infant , Molecular Targeted Therapy/methods , Neuropilin-1/immunology , Pregnancy , Pregnancy Complications, Infectious/drug therapy , Pregnancy Complications, Infectious/virology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism
14.
PLoS Pathog ; 17(12): e1010092, 2021 12.
Article in English | MEDLINE | ID: covidwho-1581718

ABSTRACT

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vesicular stomatitis Indiana virus/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , Chlorocebus aethiops , Humans , Immunization , Mice , Mice, Inbred C57BL , Mice, Transgenic , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Proteins/genetics , Viral Proteins/immunology
15.
Front Immunol ; 12: 795741, 2021.
Article in English | MEDLINE | ID: covidwho-1581316

ABSTRACT

Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired epitopes, without affecting the antigen's overall-folded structure. This study examined the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N/Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC-50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N/Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants.


Subject(s)
Antigens, Viral/immunology , COVID-19/immunology , Epitopes/immunology , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviridae/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Disease Models, Animal , Dose-Response Relationship, Immunologic , Female , Genetic Engineering , Genetic Vectors/genetics , Humans , Immunization , Mice , Neutralization Tests , Polysaccharides , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
16.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1580425

ABSTRACT

BACKGROUND: The SARS-CoV-2 spike protein mediates attachment of the virus to the host cell receptor and fusion between the virus and the cell membrane. The S1 subunit of the spike glycoprotein (S1 protein) contains the angiotensin converting enzyme 2 (ACE2) receptor binding domain. The SARS-CoV-2 variants of concern contain mutations in the S1 subunit. The spike protein is the primary target of neutralizing antibodies generated following infection, and constitutes the viral component of mRNA-based COVID-19 vaccines. METHODS: Therefore, in this work we assessed the effect of exposure (24 h) to 10 nM SARS-CoV-2 recombinant S1 protein on physiologically relevant human bronchial (bro) and alveolar (alv) lung mucosa models cultured at air-liquid interface (ALI) (n = 6 per exposure condition). Corresponding sham exposed samples served as a control. The bro-ALI model was developed using primary bronchial epithelial cells and the alv-ALI model using representative type II pneumocytes (NCI-H441). RESULTS: Exposure to S1 protein induced the surface expression of ACE2, toll like receptor (TLR) 2, and TLR4 in both bro-ALI and alv-ALI models. Transcript expression analysis identified 117 (bro-ALI) and 97 (alv-ALI) differentially regulated genes (p ≤ 0.01). Pathway analysis revealed enrichment of canonical pathways such as interferon (IFN) signaling, influenza, coronavirus, and anti-viral response in the bro-ALI. Secreted levels of interleukin (IL) 4 and IL12 were significantly (p < 0.05) increased, whereas IL6 decreased in the bro-ALI. In the case of alv-ALI, enriched terms involving p53, APRIL (a proliferation-inducing ligand) tight junction, integrin kinase, and IL1 signaling were identified. These terms are associated with lung fibrosis. Further, significantly (p < 0.05) increased levels of secreted pro-inflammatory cytokines IFNγ, IL1ꞵ, IL2, IL4, IL6, IL8, IL10, IL13, and tumor necrosis factor alpha were detected in alv-ALI, whereas IL12 was decreased. Altered levels of these cytokines are also associated with lung fibrotic response. CONCLUSIONS: In conclusion, we observed a typical anti-viral response in the bronchial model and a pro-fibrotic response in the alveolar model. The bro-ALI and alv-ALI models may serve as an easy and robust platform for assessing the pathogenicity of SARS-CoV-2 variants of concern at different lung regions.


Subject(s)
Lung/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Cytokines/metabolism , Gene Expression Profiling , Humans , Models, Biological , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism
17.
J Chem Inf Model ; 62(1): 196-209, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1574311

ABSTRACT

The angiotensin-converting enzyme II (ACE2) is a key molecular player in the regulation of vessel contraction, inflammation, and reduction of oxidative stress. In addition, ACE2 has assumed a prominent role in the fight against the COVID-19 pandemic-causing virus SARS-CoV-2, as it is the very first receptor in the host of the viral spike protein. The binding of the spike protein to ACE2 triggers a cascade of events that eventually leads the virus to enter the host cell and initiate its life cycle. At the same time, SARS-CoV-2 infection downregulates ACE2 expression especially in the lung, altering the biochemical signals regulated by the enzyme and contributing to the poor clinical prognosis characterizing the late stage of the COVID-19 disease. Despite its important biological role, a very limited number of ACE2 activators are known. Here, using a combined in silico and experimental approach, we show that ursodeoxycholic acid (UDCA) derivatives work as ACE2 activators. In detail, we have identified two potent ACE2 ligands, BAR107 and BAR708, through a docking virtual screening campaign and elucidated their mechanism of action from essential dynamics of the enzyme observed during microsecond molecular dynamics calculations. The in silico results were confirmed by in vitro pharmacological assays with the newly identified compounds showing ACE2 activity comparable to that of DIZE, the most potent ACE2 activator known so far. Our work provides structural insight into ACE2/ligand-binding interaction useful for the design of compounds with therapeutic potential against SARS-CoV-2 infection, inflammation, and other ACE2-related diseases.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2 , Antiviral Agents , Bile Acids and Salts , Humans , Pandemics , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
18.
Viruses ; 13(12)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1572669

ABSTRACT

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants' evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.


Subject(s)
Furin/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Binding Sites , Genetic Fitness , Humans , Hydrogen Bonding , Molecular Dynamics Simulation , Mutation , Phylogeny , Protein Binding , Protein Conformation , SARS-CoV-2/classification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry
19.
Viruses ; 13(12)2021 12 11.
Article in English | MEDLINE | ID: covidwho-1572661

ABSTRACT

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants' spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.


Subject(s)
Antibodies, Neutralizing/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Antigenic Variation , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Cell Fusion , Furin/metabolism , Humans , Protein Binding , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
20.
Front Immunol ; 12: 793953, 2021.
Article in English | MEDLINE | ID: covidwho-1572289

ABSTRACT

Durability of SARS-CoV-2 Spike antibody responses after infection provides information relevant to understanding protection against COVID-19 in humans. We report the results of a sequential evaluation of anti-SARS-CoV-2 antibodies in convalescent patients with a median follow-up of 14 months (range 12.4-15.4) post first symptom onset. We report persistence of antibodies for all four specificities tested [Spike, Spike Receptor Binding Domain (Spike-RBD), Nucleocapsid, Nucleocapsid RNA Binding Domain (N-RBD)]. Anti-Spike antibodies persist better than anti-Nucleocapsid antibodies. The durability analysis supports a bi-phasic antibody decay with longer half-lives of antibodies after 6 months and antibody persistence for up to 14 months. Patients infected with the Wuhan (WA1) strain maintained strong cross-reactive recognition of Alpha and Delta Spike-RBD but significantly reduced binding to Beta and Mu Spike-RBD. Sixty percent of convalescent patients with detectable WA1-specific NAb also showed strong neutralization of the Delta variant, the prevalent strain of the present pandemic. These data show that convalescent patients maintain functional antibody responses for more than one year after infection, suggesting a strong long-lasting response after symptomatic disease that may offer a prolonged protection against re-infection. One patient from this cohort showed strong increase of both Spike and Nucleocapsid antibodies at 14 months post-infection indicating SARS-CoV-2 re-exposure. These antibodies showed stronger cross-reactivity to a panel of Spike-RBD including Beta, Delta and Mu and neutralization of a panel of Spike variants including Beta and Gamma. This patient provides an example of strong anti-Spike recall immunity able to control infection at an asymptomatic level. Together, the antibodies from SARS-CoV-2 convalescent patients persist over 14 months and continue to maintain cross-reactivity to the current variants of concern and show strong functional properties.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Binding Sites, Antibody/immunology , COVID-19/virology , Cohort Studies , Cross Reactions/immunology , Female , Humans , Male , Middle Aged , Neutralization Tests/methods , Nucleocapsid/immunology , Nucleocapsid/metabolism , Protein Binding/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Time Factors
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