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1.
Biophys J ; 120(14): 2902-2913, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1605015

ABSTRACT

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 continues to rage with devastating consequences on human health and global economy. The spike glycoprotein on the surface of coronavirus mediates its entry into host cells and is the target of all current antibody design efforts to neutralize the virus. The glycan shield of the spike helps the virus to evade the human immune response by providing a thick sugar-coated barrier against any antibody. To study the dynamic motion of glycans in the spike protein, we performed microsecond-long molecular dynamics simulation in two different states that correspond to the receptor binding domain in open or closed conformations. Analysis of this microsecond-long simulation revealed a scissoring motion on the N-terminal domain of neighboring monomers in the spike trimer. The roles of multiple glycans in shielding of spike protein in different regions were uncovered by a network analysis, in which the high betweenness centrality of glycans at the apex revealed their importance and function in the glycan shield. Microdomains of glycans were identified featuring a high degree of intracommunication in these microdomains. An antibody overlap analysis revealed the glycan microdomains as well as individual glycans that inhibit access to the antibody epitopes on the spike protein. Overall, the results of this study provide detailed understanding of the spike glycan shield, which may be utilized for therapeutic efforts against this crisis.

2.
J Biomol Struct Dyn ; 39(13): 4618-4632, 2021 08.
Article in English | MEDLINE | ID: covidwho-1575345

ABSTRACT

Cinnamon has been utilized to remedy a lot of afflictions of humans. Literary works illustrate that it possesses numerous biological activities. Our research study is intended to recognize the phyto-derived antiviral substances from Cinnamon against COVID-19 main protease enzyme and to understand the in silico molecular basis of its activity. In the present study, 48 isolates compounds from Cinnamon retrieved from the PubMed database, are subjected to docking analysis. Docking study was performed using Autodock vina and PyRx software. Afterwards, admetSAR, as well as DruLiTo servers, were used to investigate drug-likeness prophecy. Our study shows that the nine phytochemicals of Cinnamon are very likely against the main protease enzyme of COVID-19. Further MD simulations could identify Tenufolin (TEN) and Pavetannin C1 (PAV) as hit compounds. Utilizing contemporary strategies, these phyto-compounds from a natural origin might establish a reliable medication or support lead identification. Identified hit compounds can be further taken for in vitro and in vivo studies to examine their effectiveness versus COVID-19.


Subject(s)
Cinnamomum zeylanicum/chemistry , Coronavirus 3C Proteases/antagonists & inhibitors , Protease Inhibitors/pharmacology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , COVID-19 , Computer Simulation , Humans , Molecular Docking Simulation , SARS-CoV-2/drug effects
3.
J Biomol Struct Dyn ; 39(15): 5768-5778, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1390294

ABSTRACT

The entire human population over the globe is currently facing appalling conditions due to the spread of infection from coronavirus disease-2019 (COVID-19). The spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present on the surface of the virion mediates the virus entry into the host cells and therefore is targeted by several scientific groups as a novel drug target site. The spike glycoprotein binds to the human angiotensin-converting enzyme-2 (hACE2) cell surface receptor abundantly expressed in lung tissues, and this binding phenomenon is a primary determinant of cell tropism and pathogenesis. The binding and internalization of the virus is the primary and most crucial step in the process of infection, and therefore the molecules targeting the inhibition of this process certainly hold a significant therapeutic value. Thus, we systematically applied the computational techniques to identify the plausible inhibitor from a chosen set of well characterized diaryl pyrimidine analogues which may disrupt interfacial interaction of spike glycoprotein (S) at the surface of hACE2. Using molecular docking, molecular dynamics (MD) simulation and binding free energy calculation, we have identified AP-NP (2-(2-amino-5-(naphthalen-2-yl)pyrimidin-4-yl)phenol), AP-3-OMe-Ph (2-(2-amino-5-(3-methoxyphenyl)pyrimidin-4-yl)phenol) and AP-4-Me-Ph (2-(2-amino-5-(p-tolyl) pyrimidin-4-yl)phenol) from a group of diaryl pyrimidine derivatives which appears to bind at the interface of the hACE2-S complex with low binding free energy. Thus, pyrimidine derivative AP-NP may be explored as an effective inhibitor for hACE2-S complex. Furthermore, in vitro and in vivo studies will strengthen the use of these inhibitors as suitable drug candidates against SARS-COV-2. Communicated by Ramaswamy H. Sarma.


Subject(s)
COVID-19 , Pharmaceutical Preparations , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Pyrimidines/pharmacology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
4.
J King Saud Univ Sci ; 33(2): 101344, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1386058

ABSTRACT

Camptothetin (CPT) is a quinoline alkaloid originally isolated from the Chinese tree, Camptotheca acuminata Decne. CPT was found to have anticancerous and antiviral properties. Derivatives of natural CPT, including topothecan and irinotecan are used clinically to treat a variety of cancers. Apart from Camptotheca acuminata Decne, CPT production was also found in the perennial plant Ophiorrhiza mungos. In this study we attempted the immobilization of the tissue culture grown callus of Ophiorrhiza mungos for the continuous production of a higher concentration of CPT. As evident from previous studies about the antiviral effects of CPT, we wanted to bioinformatically analyze the binding potential of CPT towards two important proteins of SARS-CoV-2, protease (Mpro) and RNA dependent RNA polymerase (RdRp). Further docking analysis of the CPT against the exterior spike glycoprotein of SARS-CoV-2 was also done to determine their potential interaction. The immobilized callus of Ophiorrhiza mungos produced CPT at a concentration of 420 µg/l by the end of 12 days of growth. The HPLC analysis was done to determine the purity of the CPT synthesized by the immobilization technique. The bioinformatic analysis revealed a higher binding efficiency of CPT and its derivatives, toptecan and irinotecan against Mpro and RdRp. The docking analysis of CPT against the spike glycoprotein of SARS-CoV-2 showed hydrogen bonding with the amino acids at K466 with a bond distance of 2.56A° and K355 with a bond distance of 2.40A°. This finding was of particular importance that other compounds including hydroxychloroquine sulphate, lopinavir and ivermectin could bind with the spike protein only by weak Vander wall bonds and no hydrogen bond formation was noticed. Our studies hence evaluate the efficiency of CPT against SARS-CoV-2, by potentially blocking the interaction of the spike glycoprotein with the angiotensin-converting enzyme 2 (ACE2) receptor found on host cells.

5.
Virol J ; 18(1): 1, 2021 01 04.
Article in English | MEDLINE | ID: covidwho-1388776

ABSTRACT

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.


Subject(s)
COVID-19/immunology , Leukemia/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , HEK293 Cells , Humans , Immunoglobulin G/blood , Mice
6.
Brief Bioinform ; 22(2): 1006-1022, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1387712

ABSTRACT

Interaction of SARS-CoV-2 spike glycoprotein with the ACE2 cell receptor is very crucial for virus attachment to human cells. Selected mutations in SARS-CoV-2 S-protein are reported to strengthen its binding affinity to mammalian ACE2. The N501T mutation in SARS-CoV-2-CTD furnishes better support to hotspot 353 in comparison with SARS-CoV and shows higher affinity for receptor binding. Recombination analysis exhibited higher recombination events in SARS-CoV-2 strains, irrespective of their geographical origin or hosts. Investigation further supports a common origin among SARS-CoV-2 and its predecessors, SARS-CoV and bat-SARS-like-CoV. The recombination events suggest a constant exchange of genetic material among the co-infecting viruses in possible reservoirs and human hosts before SARS-CoV-2 emerged. Furthermore, a comprehensive analysis of codon usage bias (CUB) in SARS-CoV-2 revealed significant CUB among the S-genes of different beta-coronaviruses governed majorly by natural selection and mutation pressure. Various indices of codon usage of S-genes helped in quantifying its adaptability in other animal hosts. These findings might help in identifying potential experimental animal models for investigating pathogenicity for drugs and vaccine development experiments.


Subject(s)
Biological Evolution , Codon Usage , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Humans , Models, Animal , Mutation , RNA, Transfer/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
J Proteome Res ; 19(11): 4398-4406, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-1387124

ABSTRACT

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.


Subject(s)
Aminopeptidases/metabolism , Antigens, Viral , Epitopes , Spike Glycoprotein, Coronavirus , Antigens, Viral/chemistry , Antigens, Viral/metabolism , Chromatography, Liquid , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , HLA Antigens/chemistry , HLA Antigens/metabolism , Humans , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Proteomics/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Tandem Mass Spectrometry
9.
Brief Bioinform ; 22(2): 1239-1253, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352106

ABSTRACT

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.


Subject(s)
Mutation, Missense , Spike Glycoprotein, Coronavirus/genetics , COVID-19/metabolism , COVID-19/virology , Humans , Protein Binding , SARS-CoV-2/metabolism , Thermodynamics
10.
Brief Bioinform ; 22(2): 1309-1323, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1352112

ABSTRACT

The recurrent and recent global outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has turned into a global concern which has infected more than 42 million people all over the globe, and this number is increasing in hours. Unfortunately, no vaccine or specific treatment is available, which makes it more deadly. A vaccine-informatics approach has shown significant breakthrough in peptide-based epitope mapping and opens the new horizon in vaccine development. In this study, we have identified a total of 15 antigenic peptides [including thymus cells (T-cells) and bone marrow or bursa-derived cells] in the surface glycoprotein (SG) of SARS-CoV-2 which is nontoxic and nonallergenic in nature, nonallergenic, highly antigenic and non-mutated in other SARS-CoV-2 virus strains. The population coverage analysis has found that cluster of differentiation 4 (CD4+) T-cell peptides showed higher cumulative population coverage over cluster of differentiation 8 (CD8+) peptides in the 16 different geographical regions of the world. We identified 12 peptides ((LTDEMIAQY, WTAGAAAYY, WMESEFRVY, IRASANLAA, FGAISSVLN, VKQLSSNFG, FAMQMAYRF, FGAGAALQI, YGFQPTNGVGYQ, LPDPSKPSKR, QTQTNSPRRARS and VITPGTNTSN) that are $80\hbox{--} 90\%$ identical with experimentally determined epitopes of SARS-CoV, and this will likely be beneficial for a quick progression of the vaccine design. Moreover, docking analysis suggested that the identified peptides are tightly bound in the groove of human leukocyte antigen molecules which can induce the T-cell response. Overall, this study allows us to determine potent peptide antigen targets in the SG on intuitive grounds, which opens up a new horizon in the coronavirus disease (COVID-19) research. However, this study needs experimental validation by in vitro and in vivo.


Subject(s)
COVID-19/prevention & control , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Vaccines, Subunit/immunology , Amino Acid Sequence , COVID-19/immunology , Computational Biology , Epitopes, B-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/chemistry , HLA Antigens/chemistry , Humans , Molecular Docking Simulation , Vaccines, Subunit/chemistry
11.
PLoS Pathog ; 17(6): e1009662, 2021 06.
Article in English | MEDLINE | ID: covidwho-1259253

ABSTRACT

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA's activity could be a target for anti-viral therapies.


Subject(s)
Endocytosis , RNA Viruses/immunology , Receptors, Immunologic/physiology , Virus Internalization , Animals , Antiviral Agents/pharmacology , Cell Line , Cell Membrane/virology , Chlorocebus aethiops , Drug Delivery Systems , Integrins/immunology , Interleukin-4/pharmacology , Mice , Mice, Knockout , Protein Domains , Receptors, Immunologic/genetics , Vero Cells
12.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1255762

ABSTRACT

BACKGROUNDRecent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2.METHODSWe used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system.RESULTSMemory CD4+ T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to monospecific CD4+ T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC.CONCLUSIONSOur data confirm, for what we believe is the first time, the existence of unique memory CD4+ T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients.FUNDINGNIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Adult , Aged , Cross Reactions , Female , Humans , Jurkat Cells , Male , Middle Aged
13.
Biochem Biophys Res Commun ; 565: 8-13, 2021 08 06.
Article in English | MEDLINE | ID: covidwho-1252489

ABSTRACT

Amidst infectious disease outbreaks, a practical tool that can quantitatively monitor individuals' antibodies to pathogens is vital for disease control. The currently used serological lateral flow immunoassays (LFIAs) can only detect the presence of antibodies for a single antigen. Here, we fabricated a multiplexed circular flow immunoassay (CFIA) test strip with YOLO v4-based object recognition that can quickly quantify and differentiate antibodies that bind membrane glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or hemagglutinin of influenza A (H1N1) virus in the sera of immunized mice in one assay using one sample. Spot intensities were found to be indicative of antibody titers to membrane glycoprotein of SARS-CoV-2 and were, thus, quantified relative to spots from immunoglobulin G (IgG) reaction in a CFIA to account for image heterogeneity. Quantitative intensities can be displayed in real time alongside an image of CFIA that was captured by a built-in camera. We demonstrate for the first time that CFIA is a specific, multi-target, and quantitative tool that holds potential for digital and simultaneous monitoring of antibodies recognizing various pathogens including SARS-CoV-2.


Subject(s)
Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/immunology , Coronavirus M Proteins/immunology , Immunoassay/methods , SARS-CoV-2/immunology , Animals , COVID-19/virology , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , SARS-CoV-2/isolation & purification
14.
Front Mol Biosci ; 8: 637989, 2021.
Article in English | MEDLINE | ID: covidwho-1247881

ABSTRACT

This work investigated the interaction of indole with SARS-CoV-2. Indole is widely used as a medical material owing to its astounding biological activities. Indole and its derivatives belong to a significant category of heterocyclic compounds that have been used as a crucial component for several syntheses of medicine. A straightforward one-pot three-component synthesis of indole, coupled with Mannich base derivatives 1a-1j, was synthesized without a catalyst. The products were confirmed by IR, 1H-NMR, 13C-NMR, mass spectra, and elemental analysis. The indole derivatives were tested for cytotoxic activity, using three cancer cell lines and normal cell lines of Human embryonic kidney cell (HEK293), liver cell (LO2), and lung cell (MRC5) by MTT assay using doxorubicin as the standard drug. The result of cytotoxicity indole compound 1c (HepG2, LC50-0.9 µm, MCF-7, LC50-0.55 µm, HeLa, LC50-0.50 µm) was found to have high activity compared with other compounds used for the same purpose. The synthesized derivatives have revealed their safety by exhibiting significantly less cytotoxicity against the normal cell line (HEK-293), (LO2), and (MRC5) with IC50 > 100 µg/ml. Besides, we report an in silico study with spike glycoprotein (SARS-CoV-2-S). The selective molecules of compound 1c exhibited the highest docking score -2.808 (kcal/mol) compared to other compounds. This research work was successful in synthesizing a few compounds with potential as anticancer agents. Furthermore, we have tried to emphasize the anticipated role of indole scaffolds in designing and discovering the much-awaited anti-SARS CoV-2 therapy by exploring the research articles depicting indole moieties as targeting SARS CoV-2 coronavirus.

15.
Front Immunol ; 12: 641360, 2021.
Article in English | MEDLINE | ID: covidwho-1247859

ABSTRACT

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models.


Subject(s)
COVID-19/prevention & control , Influenza A virus/physiology , Pulmonary Surfactant-Associated Protein D/metabolism , Respiratory Mucosa/physiology , Respiratory Syncytial Viruses/physiology , Virion/metabolism , Angiotensin-Converting Enzyme 2/metabolism , HEK293 Cells , Humans , Immunity, Innate , Protein Binding , Pulmonary Surfactant-Associated Protein D/genetics , Recombinant Proteins/genetics , Respiratory Mucosa/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
16.
J Virol ; 95(15): e0049621, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1243690

ABSTRACT

The Severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 originated in bats and adapted to infect humans. Several SARS-CoV-2 strains have been identified. Genetic variation is fundamental to virus evolution and, in response to selection pressure, is manifested as the emergence of new strains and species adapted to different hosts or with novel pathogenicity. The combination of variation and selection forms a genetic footprint on the genome, consisting of the preferential accumulation of mutations in particular areas. Properties of betacoronaviruses contributing to variation and the emergence of new strains and species are beginning to be elucidated. To better understand their variation, we profiled the accumulation of mutations in all species in the genus Betacoronavirus, including SARS-CoV-2 and two other species that infect humans: SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Variation profiles identified both genetically stable and variable areas at homologous locations across species within the genus Betacoronavirus. The S glycoprotein is the most variable part of the genome and is structurally disordered. Other variable parts include proteins 3 and 7 and ORF8, which participate in replication and suppression of antiviral defense. In contrast, replication proteins in ORF1b are the least variable. Collectively, our results show that variation and structural disorder in the S glycoprotein is a general feature of all members of the genus Betacoronavirus, including SARS-CoV-2. These findings highlight the potential for the continual emergence of new species and strains with novel biological properties and indicate that the S glycoprotein has a critical role in host adaptation. IMPORTANCE Natural infection with SARS-CoV-2 and vaccines triggers the formation of antibodies against the S glycoprotein, which are detected by antibody-based diagnostic tests. Our analysis showed that variation in the S glycoprotein is a general feature of all species in the genus Betacoronavirus, including three species that infect humans: SARS-CoV, SARS-CoV-2, and MERS-CoV. The variable nature of the S glycoprotein provides an explanation for the emergence of SARS-CoV-2, the differentiation of SARS-CoV-2 into strains, and the probability of SARS-CoV-2 repeated infections in people. Variation of the S glycoprotein also has important implications for the reliability of SARS-CoV-2 antibody-based diagnostic tests and the design and deployment of vaccines and antiviral drugs. These findings indicate that adjustments to vaccine design and deployment and to antibody-based diagnostic tests are necessary to account for S glycoprotein variation.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Genetic Variation , Genome, Viral , Spike Glycoprotein, Coronavirus/genetics , Genome-Wide Association Study , Humans
17.
Comput Struct Biotechnol J ; 19: 3187-3197, 2021.
Article in English | MEDLINE | ID: covidwho-1242911

ABSTRACT

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pathological pulmonary symptoms. Most efforts to develop vaccines and drugs against this virus target the spike glycoprotein, particularly its S1 subunit, which is recognised by angiotensin-converting enzyme 2. Here we use the in-house developed tool CaverDock to perform virtual screening against spike glycoprotein using a cryogenic electron microscopy structure (PDB-ID: 6VXX) and the representative structures of five most populated clusters from a previously published molecular dynamics simulation. The dataset of ligands was obtained from the ZINC database and consists of drugs approved for clinical use worldwide. Trajectories for the passage of individual drugs through the tunnel of the spike glycoprotein homotrimer, their binding energies within the tunnel, and the duration of their contacts with the trimer's three subunits were computed for the full dataset. Multivariate statistical methods were then used to establish structure-activity relationships and select top candidate for movement inhibition. This new protocol for the rapid screening of globally approved drugs (4359 ligands) in a multi-state protein structure (6 states) showed high robustness in the rate of finished calculations. The protocol is universal and can be applied to any target protein with an experimental tertiary structure containing protein tunnels or channels. The protocol will be implemented in the next version of CaverWeb (https://loschmidt.chemi.muni.cz/caverweb/) to make it accessible to the wider scientific community.

18.
J Clin Invest ; 131(10)2021 05 17.
Article in English | MEDLINE | ID: covidwho-1238630

ABSTRACT

BACKGROUNDRecent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2.METHODSWe used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system.RESULTSMemory CD4+ T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to monospecific CD4+ T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC.CONCLUSIONSOur data confirm, for what we believe is the first time, the existence of unique memory CD4+ T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients.FUNDINGNIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , Immunologic Memory , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Adult , Aged , Cross Reactions , Female , Humans , Jurkat Cells , Male , Middle Aged
19.
MMWR Morb Mortal Wkly Rep ; 70(20): 749-752, 2021 May 21.
Article in English | MEDLINE | ID: covidwho-1237004

ABSTRACT

The Pfizer-BioNTech COVID-19 (BNT162b2) vaccine is a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine encoding the prefusion spike glycoprotein of SARS-CoV-2, the virus that causes COVID-19. Vaccination with the Pfizer-BioNTech COVID-19 vaccine consists of 2 intramuscular doses (30 µg, 0.3 mL each) administered 3 weeks apart. On December 11, 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for use of the Pfizer-BioNTech COVID-19 vaccine (Pfizer, Inc; Philadelphia, Pennsylvania) in persons aged ≥16 years (1); on December 12, 2020, the Advisory Committee on Immunization Practices (ACIP) issued an interim recommendation for use of the vaccine in the same age group (2). As of May 12, 2021, approximately 141.6 million doses of the Pfizer-BioNTech COVID-19 vaccine had been administered to persons aged ≥16 years.* On May 10, 2021, FDA expanded the EUA for the Pfizer-BioNTech COVID-19 vaccine to include adolescents aged 12-15 years (1). On May 12, 2021, ACIP issued an interim recommendation† for use of the Pfizer-BioNTech COVID-19 vaccine in adolescents aged 12-15 years for the prevention of COVID-19. To guide its deliberations regarding the vaccine, ACIP used the Evidence to Recommendation (EtR) Framework,§ using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach.¶ The ACIP recommendation for the use of the Pfizer-BioNTech COVID-19 vaccine in persons aged ≥12 years under an EUA is interim and will be updated as additional information becomes available.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunization/standards , Practice Guidelines as Topic , Adolescent , Advisory Committees , COVID-19/epidemiology , Child , Drug Approval , Humans , United States/epidemiology
20.
J Biomol Struct Dyn ; : 1-28, 2021 May 20.
Article in English | MEDLINE | ID: covidwho-1236146

ABSTRACT

The COVID-19 pandemic has severely destructed human life worldwide, with no suitable treatment until now. SARS-CoV-2 virus is unprecedented, resistance against number of therapeutics and spreading rapidly with high mortality, which warrants the need to discover new effective drugs to combat this situation. This current study is undertaken to explore the antiviral potential of marine algal compounds to inhibit the viral entry and its multiplication using computational analysis. Among the proven drug discovery targets of SARS-CoV-2, spike glycoprotein and 3-chymotrypsin-like protease are responsible for the virus attachment and viral genome replication in the host cell. In this study, the above-mentioned drug targets were docked with marine algal compounds (sulfated polysaccharides, polysaccharide derivatives and polyphenols) using molecular docking tools (AutoDockTools). The obtained results indicate that κ-carrageenan, laminarin, eckol, trifucol and ß-D-galactose are the top-ranking compounds showing better docking scores with SARS-CoV-2 targets, than the current experimental COVID-19 antiviral drugs like dexamethasone, remdesivir, favipiravir and MIV-150. Further, molecular dynamic simulation, ADMET and density functional theory calculations were evaluated to substantiate the findings. To the best of our knowledge, this is the first report on in silico analysis of aforesaid algal metabolites against SARS-CoV-2 targets. This study concludes that these metabolites can be curative for COVID-19 in the hour of need after further validations in in vitro and in vivo testings.Communicated by Ramaswamy H. Sarma.

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