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1.
Proc Natl Acad Sci U S A ; 119(8)2022 02 22.
Article in English | MEDLINE | ID: covidwho-1671749

ABSTRACT

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2-infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2.


Subject(s)
COVID-19/drug therapy , Interferon-alpha/pharmacology , SARS-CoV-2/drug effects , Transcriptome , Virus Replication/drug effects , Animals , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Cloning, Molecular , Disease Models, Animal , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Profiling , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Mice , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/pharmacology , Recombinant Proteins/classification , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Signal Transduction , Vero Cells
2.
Cell ; 185(5): 896-915.e19, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1670278

ABSTRACT

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunity, Mucosal , Administration, Intranasal , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , COVID-19/virology , COVID-19 Vaccines/immunology , Cytokines/blood , Genetic Vectors/genetics , Genetic Vectors/immunology , Genetic Vectors/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutralization Tests , Nucleocapsid/genetics , Nucleocapsid/immunology , Nucleocapsid/metabolism , Pan troglodytes , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
3.
Biomed Pharmacother ; 146: 112527, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1559074

ABSTRACT

Coronavirus disease 2019 (COVID-19) has a devastating impact on global populations triggered by a highly infectious viral sickness, produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The third major cause of mortality in the United States, following heart disease and cancer in 2020, was undoubtedly COVID-19. The centers for disease control and prevention (CDC) and the world health organization (WHO) separately developed a categorization system for differentiating new strains of SARS-CoV-2 into variants of concern (VoCs) and variants of interest (VoIs) with the continuing development of various strains SARS-CoV-2. By December 2021, five of the SARS-CoV-2 VoCs were discovered from the onset of the pandemic depending on the latest epidemiologic report by the WHO: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Mutations in the receptor-binding domain (RBD) and n-terminal domain (NTD) have been found throughout all five identified VoCs. All strains other than the delta mutant are often found with the N501Y mutation situated on the RBD, resulting in higher binding between the spike protein and angiotensin-converting enzyme 2 (ACE2) receptors, enhanced viral adhesion, and following the entrance to host cells. The introduction of these new strains of SRAS-CoV-2 is likely to overcome the remarkable achievements gained in restricting this viral disease to the point where it is presented with remarkable vaccine developments against COVID-19 and strong worldwide mass immunization initiatives. Throughout this literature review, the effectiveness of current COVID-19 vaccines for managing and prohibiting SARS-CoV-2 strains is thoroughly described.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Genetic Vectors/administration & dosage , SARS-CoV-2/drug effects , Vaccines, Synthetic/administration & dosage , /administration & dosage , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/genetics , COVID-19/metabolism , COVID-19 Vaccines/genetics , COVID-19 Vaccines/metabolism , Genetic Variation/genetics , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Treatment Outcome , Vaccines, Synthetic/genetics , Vaccines, Synthetic/metabolism , /metabolism
4.
Adv Sci (Weinh) ; 8(23): e2103266, 2021 12.
Article in English | MEDLINE | ID: covidwho-1479368

ABSTRACT

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.


Subject(s)
Blood Coagulation/physiology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies/chemistry , Antibodies/immunology , Antibodies/metabolism , COVID-19/diagnosis , COVID-19/virology , Endothelial Cells/chemistry , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fibrin/chemistry , Fibrin/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Interleukin-6/immunology , Liposomes/chemistry , Microfluidics/methods , Mutation , Nanoparticles/chemistry , Platelet Aggregation , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics
6.
Viruses ; 13(8)2021 07 28.
Article in English | MEDLINE | ID: covidwho-1376988

ABSTRACT

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.


Subject(s)
Adenoviruses, Human/genetics , Genetic Therapy/methods , Genetic Vectors/genetics , Adenoviruses, Human/classification , Adenoviruses, Human/metabolism , Animals , Cell Line , Genes, Reporter , Genetic Therapy/instrumentation , Genetic Vectors/metabolism , Humans , Liver/virology , Lung/virology , Mice , Phylogeny , Spleen/virology , Transduction, Genetic
7.
SLAS Discov ; 26(6): 757-765, 2021 07.
Article in English | MEDLINE | ID: covidwho-1194439

ABSTRACT

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Subject(s)
Adenosine/analogs & derivatives , High-Throughput Screening Assays , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Adenosine/chemistry , Adenosine/pharmacology , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Methylation , Methyltransferases , Models, Molecular , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Tritium , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/metabolism
8.
SLAS Discov ; 26(6): 766-774, 2021 07.
Article in English | MEDLINE | ID: covidwho-1192708

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.


Subject(s)
Antiviral Agents/pharmacology , Enzyme Inhibitors/pharmacology , Exonucleases/antagonists & inhibitors , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Enzyme Assays , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Exonucleases/genetics , Exonucleases/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
9.
J Biol Chem ; 296: 100306, 2021.
Article in English | MEDLINE | ID: covidwho-1152462

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Clathrin Heavy Chains/genetics , Endocytosis/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Clathrin Heavy Chains/antagonists & inhibitors , Clathrin Heavy Chains/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , Hydrazones/pharmacology , Lentivirus/genetics , Lentivirus/metabolism , Protein Binding/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Sulfonamides/pharmacology , Thiazolidines/pharmacology , Vero Cells
10.
SLAS Discov ; 26(6): 749-756, 2021 07.
Article in English | MEDLINE | ID: covidwho-1136206

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Subject(s)
Antiviral Agents/pharmacology , Exoribonucleases/antagonists & inhibitors , High-Throughput Screening Assays , Nitro Compounds/pharmacology , RNA Caps/antagonists & inhibitors , RNA, Viral/antagonists & inhibitors , SARS-CoV-2/drug effects , Thiazoles/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacology , Antiviral Agents/chemistry , COVID-19/virology , Cloning, Molecular , Drug Repositioning , Enzyme Assays , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Escherichia coli/metabolism , Exoribonucleases/genetics , Exoribonucleases/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Mass Spectrometry/methods , Methylation , Nitro Compounds/chemistry , Prescription Drugs/chemistry , Prescription Drugs/pharmacology , RNA Caps/genetics , RNA Caps/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Thiazoles/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
11.
J Biol Chem ; 296: 100449, 2021.
Article in English | MEDLINE | ID: covidwho-1091794

ABSTRACT

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.


Subject(s)
Curcumin/pharmacology , Drug Design , Epithelial Cells/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-hck/antagonists & inhibitors , Animals , Cell Line, Tumor , Cloning, Molecular , Curcumin/analogs & derivatives , Curcumin/chemical synthesis , Drug Stability , Epithelial Cells/enzymology , Epithelial Cells/pathology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HT29 Cells , Humans , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/chemical synthesis , /metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-hck/chemistry , Proto-Oncogene Proteins c-hck/genetics , Proto-Oncogene Proteins c-hck/metabolism , RAW 264.7 Cells , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , src-Family Kinases/genetics , src-Family Kinases/metabolism
12.
Virology ; 557: 15-22, 2021 05.
Article in English | MEDLINE | ID: covidwho-1071993

ABSTRACT

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin G/blood , SARS-CoV-2/immunology , Amino Acid Sequence , COVID-19/blood , COVID-19/immunology , COVID-19 Serological Testing/methods , Case-Control Studies , Cloning, Molecular , Coronavirus Nucleocapsid Proteins/genetics , Enzyme-Linked Immunosorbent Assay/standards , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Immune Sera/chemistry , Immunoglobulin M/blood , Phosphoproteins/genetics , Phosphoproteins/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity
13.
J Biol Chem ; 296: 100346, 2021.
Article in English | MEDLINE | ID: covidwho-1056842

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies suitable for molecular pathology research is not fully addressed. Here, we produced six mouse anti-SARS-CoV-2 spike monoclonal antibodies that not only exhibit robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also demonstrate neutralizing activity against SARS-CoV-2 infection to VeroE6/TMPRSS2 cells. Due to their mouse origin, our monoclonal antibodies are compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, providing a useful tool for future research. Furthermore, in the hope of applying the antibodies of clinical setting, we determined the variable regions of the antibodies and used them to produce recombinant human/mouse chimeric antibodies.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/prevention & control , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/chemistry , Antibodies, Viral/isolation & purification , Binding Sites , COVID-19/immunology , COVID-19/virology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Mice , Neutralization Tests , Protein Binding , Protein Interaction Domains and Motifs , Protein Subunits/administration & dosage , Protein Subunits/genetics , Protein Subunits/immunology , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/immunology , Vaccination
14.
Commun Biol ; 4(1): 129, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1054066

ABSTRACT

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Adult , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , Convalescence , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Female , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Immune Sera/chemistry , Immunity, Humoral , Lentivirus/genetics , Lentivirus/immunology , Male , Middle Aged , Neutralization Tests , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/metabolism , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Severity of Illness Index , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Survival Analysis
16.
Hum Vaccin Immunother ; 16(12): 2905-2912, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-970085

ABSTRACT

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation. Considering the crucial role of SARS-CoV-2 spike (S) glycoprotein in virus attachment, entry, and induction of neutralizing antibodies, S protein is being widely used as a target for vaccine development. Based on advances in techniques for vaccine design, inactivated, live-vectored, nucleic acid, and recombinant COVID-19 vaccines are being developed and tested for their efficacy. Phase3 clinical trials are underway or will soon begin for several of these vaccines. Assuming that clinical efficacy is shown for one or more vaccines, safety is a major aspect to be considered before deploying such vaccines to the public. The current review focuses on the recent advances in recombinant COVID-19 vaccine research and development and associated issues.


Subject(s)
COVID-19 Vaccines/therapeutic use , COVID-19/prevention & control , Vaccines, Synthetic/therapeutic use , COVID-19/genetics , COVID-19/metabolism , COVID-19 Vaccines/genetics , COVID-19 Vaccines/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Genetic Vectors/therapeutic use , Humans , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Synthetic/metabolism
17.
Viruses ; 12(7)2020 06 28.
Article in English | MEDLINE | ID: covidwho-683587

ABSTRACT

Viral vectored vaccines are desirable alternatives for conventional infectious bronchitis virus (IBV) vaccines. We have recently shown that a recombinant Newcastle disease virus (rNDV) strain LaSota expressing the spike (S) protein of IBV strain Mass-41 (rLaSota/IBV-S) was a promising vaccine candidate for IBV. Here we evaluated a novel chimeric rNDV/avian paramyxovirus serotype 2 (rNDV/APMV-2) as a vaccine vector against IBV. The rNDV/APMV-2 vector was chosen because it is much safer than the rNDV strain LaSota vector, particularly for young chicks and chicken embryos. In order to determine the effectiveness of this vector, a recombinant rNDV/APMV-2 expressing the S protein of IBV strain Mass-41 (rNDV/APMV-2/IBV-S) was constructed. The protective efficacy of this vector vaccine was compared to that of the rNDV vector vaccine. In one study, groups of one-day-old specific-pathogenic-free (SPF) chickens were immunized with rLaSota/IBV-S and rNDV/APMV-2/IBV-S and challenged four weeks later with the homologous highly virulent IBV strain Mass-41. In another study, groups of broiler chickens were single (at day one or three weeks of age) or prime-boost (prime at day one and boost at three weeks of age) immunized with rLaSota/IBV-S and/or rNDV-APMV-2/IBV-S. At weeks six of age, chickens were challenged with a highly virulent IBV strain Mass-41. Our challenge study showed that novel rNDV/APMV-2/IBV-S provided similar protection as rLaSota/IBV-S in SPF chickens. However, compared to prime-boost immunization of chickens with chimeric rNDV/APMV-2, rLaSota/IBV-S and/or a live IBV vaccine, single immunization of chickens with rLaSota/IBV-S, or live IBV vaccine provided better protection against IBV. In conclusion, we have developed the novel rNDV/APMV-2 vector expressing S protein of IBV that can be a safer vaccine against IB in chickens. Our results also suggest a single immunization with a LaSota vectored IBV vaccine candidate provides better protection than prime-boost immunization regimens.


Subject(s)
Avulavirus/genetics , Coronavirus Infections/veterinary , Genetic Vectors/genetics , Infectious bronchitis virus/immunology , Poultry Diseases/prevention & control , Viral Vaccines/administration & dosage , Animals , Avulavirus/metabolism , Chickens , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Genetic Vectors/metabolism , Infectious bronchitis virus/genetics , Infectious bronchitis virus/pathogenicity , Poultry Diseases/virology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Proteins/administration & dosage , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology
18.
Protein Sci ; 29(7): 1596-1605, 2020 07.
Article in English | MEDLINE | ID: covidwho-71902

ABSTRACT

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.


Subject(s)
Betacoronavirus/chemistry , Endoribonucleases/chemistry , Middle East Respiratory Syndrome Coronavirus/chemistry , Oligonucleotides/chemistry , SARS Virus/chemistry , Viral Nonstructural Proteins/chemistry , Amino Acid Sequence , Betacoronavirus/genetics , Betacoronavirus/metabolism , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Endoribonucleases/genetics , Endoribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Models, Molecular , Oligonucleotides/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
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