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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
3.
Hum Gene Ther ; 32(11-12): 541-562, 2021 06.
Article in English | MEDLINE | ID: covidwho-1216585

ABSTRACT

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease (COVID-19) caused by the novel coronavirus SARS-coronavirus 2 (CoV-2). To combat the devastating spread of SARS-CoV-2, extraordinary efforts from numerous laboratories have focused on the development of effective and safe vaccines. Traditional live-attenuated or inactivated viral vaccines are not recommended for immunocompromised patients as the attenuated virus can still cause disease via phenotypic or genotypic reversion. Subunit vaccines require repeated dosing and adjuvant use to be effective, and DNA vaccines exhibit lower immune responses. mRNA vaccines can be highly unstable under physiological conditions. On the contrary, naturally antigenic viral vectors with well-characterized structure and safety profile serve as among the most effective gene carriers to provoke immune response via heterologous gene transfer. Viral vector-based vaccines induce both an effective cellular immune response and a humoral immune response owing to their natural adjuvant properties via transduction of immune cells. Consequently, viral vectored vaccines carrying the SARS-CoV-2 spike protein have recently been generated and successfully used to activate cytotoxic T cells and develop a neutralizing antibody response. Recent progress in SARS-CoV-2 vaccines, with an emphasis on gene therapy viral vector-based vaccine development, is discussed in this review.


Subject(s)
COVID-19 Vaccines/pharmacology , Genetic Vectors , Vaccines, Attenuated/pharmacology , Vaccines, Synthetic/pharmacology , Viral Structural Proteins/chemistry , Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors/chemistry , Genetic Vectors/genetics , Humans , Lentivirus/genetics , SARS-CoV-2/genetics , Vaccines, DNA/pharmacology , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolism
4.
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
5.
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
6.
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
7.
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
8.
Virology ; 557: 70-85, 2021 05.
Article in English | MEDLINE | ID: covidwho-1108783

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into the human population in late 2019 and caused the global COVID-19 pandemic. SARS-CoV-2 has spread to more than 215 countries and infected many millions of people. Despite the introduction of numerous governmental and public health measures to control disease spread, infections continue at an unabated pace, suggesting that effective vaccines and antiviral drugs will be required to curtail disease, end the pandemic, and restore societal norms. Here, we review the current developments in antibody and vaccine countermeasures to limit or prevent disease.


Subject(s)
Antibodies, Viral/biosynthesis , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Pandemics , SARS-CoV-2/immunology , Animals , COVID-19/epidemiology , COVID-19/immunology , COVID-19/therapy , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Clinical Trials as Topic , Disease Models, Animal , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunity, Innate/drug effects , Immunization, Passive/methods , Immunogenicity, Vaccine , Patient Safety , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/biosynthesis , Vaccines, Virus-Like Particle/immunology
9.
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
10.
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
11.
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
12.
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
13.
Vaccine ; 39(2): 197-201, 2021 01 08.
Article in English | MEDLINE | ID: covidwho-960152

ABSTRACT

The threat of the current coronavirus disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is accelerating the development of potential vaccines. Candidate vaccines have been generated using existing technologies that have been applied for developing vaccines against other infectious diseases. Two new types of platforms, mRNA- and viral vector-based vaccines, have been gaining attention owing to the rapid advancement in their methodologies. In clinical trials, setting appropriate immunological endpoints plays a key role in evaluating the efficacy and safety of candidate vaccines. Updated information about immunological features from individuals who have or have not been exposed to SARS-CoV-2 continues to guide effective vaccine development strategies. This review highlights key strategies for generating candidate SARS-CoV-2 vaccines and considerations for vaccine development and clinical trials.


Subject(s)
Antibodies, Viral/biosynthesis , COVID-19 Vaccines/biosynthesis , COVID-19/prevention & control , Pandemics/prevention & control , SARS-CoV-2/immunology , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/prevention & control , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Clinical Trials as Topic , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunity, Innate/drug effects , Immunization Schedule , Immunogenicity, Vaccine , Patient Safety , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit , Vaccines, Virus-Like Particle
14.
Rev Med Virol ; 31(3): e2183, 2021 05.
Article in English | MEDLINE | ID: covidwho-864731

ABSTRACT

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which is responsible for a global pandemic that started in late 2019 in Wuhan, China. To prevent the worldwide spread of this highly pathogenic virus, development of an effective and safe vaccine is urgently needed. The SARS-CoV-2 and SARS-CoV share a high degree of genetic and pathologic identity and share safety and immune-enhancement concerns regarding vaccine development. Prior animal studies with first generation (whole virus-based) preparations of SARS-CoV vaccines (inactivated and attenuated vaccine modalities) indicated the possibility of increased infectivity or eosinophilic infiltration by immunization. Therefore, development of second and third generation safer vaccines (by using modern vaccine platforms) is actively sought for this viral infection. The spike (S) protein of SARS-CoVs is the main determinant of cell entry and tropism and is responsible for facilitating zoonosis into humans and sustained person-to-person transmission. Furthermore, 'S' protein contains multiple neutralizing epitopes that play an essential role in the induction of neutralizing antibodies (nAbs) and protective immunity. Moreover, T-cell responses against the SARS-CoV-2 'S' protein have also been characterized that correlate to the IgG and IgA antibody titres in Covid-19 patients. Thus, S protein is an obvious candidate antigen for inclusion into vaccine platforms against SARS-CoV-2 viral infection. This manuscript reviews different characteristics of S protein, its potency and 'state of the art' of the vaccine development strategies and platforms using this antigen, for construction of a safe and effective SARS-CoV-2 vaccine.


Subject(s)
Antibodies, Viral/biosynthesis , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Genome, Viral/immunology , Pandemics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Clinical Trials as Topic , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunity, Innate/drug effects , Immunization Schedule , Immunogenicity, Vaccine , Patient Safety , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit
15.
J Med Virol ; 92(10): 2114-2123, 2020 10.
Article in English | MEDLINE | ID: covidwho-777547

ABSTRACT

The present study provides the first multiepitope vaccine construct using the 3CL hydrolase protein of SARS-CoV-2. The coronavirus 3CL hydrolase (Mpro) enzyme is essential for proteolytic maturation of the virus. This study was based on immunoinformatics and structural vaccinology strategies. The design of the multiepitope vaccine was built using helper T-cell and cytotoxic T-cell epitopes from the 3CL hydrolase protein along with an adjuvant to enhance immune response; these are joined to each other by short peptide linkers. The vaccine also carries potential B-cell linear epitope regions, B-cell discontinuous epitopes, and interferon-γ-inducing epitopes. Epitopes of the constructed multiepitope vaccine were found to be antigenic, nonallergic, nontoxic, and covering large human populations worldwide. The vaccine construct was modeled, validated, and refined by different programs to achieve a high-quality three-dimensional structure. The resulting high-quality model was applied for conformational B-cell epitope selection and docking analyses with toll-like receptor-3 for understanding the capability of the vaccine to elicit an immune response. In silico cloning and codon adaptation were also performed with the pET-19b plasmid vector. The designed multiepitope peptide vaccine may prompt the development of a vaccine to control SARS-CoV-2 infection.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus 3C Proteases/immunology , Epitopes, B-Lymphocyte/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Amino Acid Sequence , Binding Sites , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , Cloning, Molecular/methods , Computational Biology/methods , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Genetic Vectors/chemistry , Genetic Vectors/immunology , HLA Antigens/chemistry , HLA Antigens/genetics , HLA Antigens/immunology , Humans , Immunity, Innate/drug effects , Immunogenicity, Vaccine , Interferon-gamma/genetics , Interferon-gamma/immunology , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , T-Lymphocytes, Cytotoxic/chemistry , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/virology , T-Lymphocytes, Helper-Inducer/chemistry , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/virology , Toll-Like Receptor 3/chemistry , Toll-Like Receptor 3/genetics , User-Computer Interface , Vaccines, Subunit
16.
Nat Rev Immunol ; 20(10): 615-632, 2020 10.
Article in English | MEDLINE | ID: covidwho-744378

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most formidable challenge to humanity in a century. It is widely believed that prepandemic normalcy will never return until a safe and effective vaccine strategy becomes available and a global vaccination programme is implemented successfully. Here, we discuss the immunological principles that need to be taken into consideration in the development of COVID-19 vaccine strategies. On the basis of these principles, we examine the current COVID-19 vaccine candidates, their strengths and potential shortfalls, and make inferences about their chances of success. Finally, we discuss the scientific and practical challenges that will be faced in the process of developing a successful vaccine and the ways in which COVID-19 vaccine strategies may evolve over the next few years.


Subject(s)
Antibodies, Viral/biosynthesis , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/prevention & control , Viral Vaccines/immunology , Betacoronavirus/drug effects , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Clinical Trials as Topic , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunity, Herd/drug effects , Immunity, Innate/drug effects , Immunization Schedule , Immunogenicity, Vaccine , Patient Safety , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS-CoV-2 , Severe Acute Respiratory Syndrome/immunology , Severe Acute Respiratory Syndrome/virology , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit , Vaccines, Virus-Like Particle , Viral Vaccines/administration & dosage , Viral Vaccines/biosynthesis
17.
Virus Res ; 288: 198141, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-725473

ABSTRACT

The recent outbreak of the betacoronavirus SARS-CoV-2 has become a significant concern to public health care worldwide. As of August 19, 2020, more than 22,140,472 people are infected, and over 781,135 people have died due to this deadly virus. In the USA alone, over 5,482,602 people are currently infected, and more than 171,823 people have died. SARS-CoV-2 has shown a higher infectivity rate and a more extended incubation period as compared to previous coronaviruses. SARS-CoV-2 binds much more strongly than SARS-CoV to the same host receptor, angiotensin-converting enzyme 2 (ACE2). Previously, several methods to develop a vaccine against SARS-CoV or MERS-CoV have been tried with limited success. Since SARS-CoV-2 uses the spike (S) protein for entry to the host cell, it is one of the most preferred targets for making vaccines or therapeutics against SARS-CoV-2. In this review, we have summarised the characteristics of the S protein, as well as the different approaches being used for the development of vaccines and/or therapeutics based on the S protein.


Subject(s)
Antibodies, Viral/biosynthesis , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Angiotensin-Converting Enzyme 2 , Antibody-Dependent Enhancement/drug effects , Betacoronavirus/drug effects , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Clinical Trials as Topic , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunogenicity, Vaccine , Patient Safety , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/immunology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/biosynthesis , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/biosynthesis
18.
Virus Res ; 288: 198114, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-709007

ABSTRACT

The current COVID-19 pandemic has urged the scientific community internationally to find answers in terms of therapeutics and vaccines to control SARS-CoV-2. Published investigations mostly on SARS-CoV and to some extent on MERS has taught lessons on vaccination strategies to this novel coronavirus. This is attributed to the fact that SARS-CoV-2 uses the same receptor as SARS-CoV on the host cell i.e. human Angiotensin Converting Enzyme 2 (hACE2) and is approximately 79% similar genetically to SARS-CoV. Though the efforts on COVID-19 vaccines started very early, initially in China, as soon as the outbreak of novel coronavirus erupted and then world-over as the disease was declared a pandemic by WHO. But we will not be having an effective COVID-19 vaccine before September, 2020 as per very optimistic estimates. This is because a successful COVID-19 vaccine will require a cautious validation of efficacy and adverse reactivity as the target vaccinee population include high-risk individuals over the age of 60, particularly those with chronic co-morbid conditions, frontline healthcare workers and those involved in essentials industries. Various platforms for vaccine development are available namely: virus vectored vaccines, protein subunit vaccines, genetic vaccines, and monoclonal antibodies for passive immunization which are under evaluations for SARS-CoV-2, with each having discrete benefits and hindrances. The COVID-19 pandemic which probably is the most devastating one in the last 100 years after Spanish flu mandates the speedy evaluation of the multiple approaches for competence to elicit protective immunity and safety to curtail unwanted immune-potentiation which plays an important role in the pathogenesis of this virus. This review is aimed at providing an overview of the efforts dedicated to an effective vaccine for this novel coronavirus which has crippled the world in terms of economy, human health and life.


Subject(s)
Antibodies, Viral/biosynthesis , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Angiotensin-Converting Enzyme 2 , Betacoronavirus/drug effects , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Clinical Trials as Topic , Coronavirus Infections/epidemiology , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Coronavirus Infections/virology , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Immunity, Innate/drug effects , Immunization, Passive/methods , Immunogenicity, Vaccine , Patient Safety , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/immunology , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/epidemiology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, Virus/genetics , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2 , Vaccines, Attenuated , Vaccines, DNA , Vaccines, Subunit , Vaccines, Virus-Like Particle/administration & dosage , Vaccines, Virus-Like Particle/biosynthesis , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/administration & dosage , Viral Vaccines/biosynthesis
19.
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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