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1.
Int J Mol Sci ; 23(4)2022 Feb 16.
Article in English | MEDLINE | ID: covidwho-1708485

ABSTRACT

Despite the fact that a range of vaccines against COVID-19 have already been created and are used for mass vaccination, the development of effective, safe, technological, and affordable vaccines continues. We have designed a vaccine that combines the recombinant protein and DNA vaccine approaches in a self-assembled particle. The receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 was conjugated to polyglucin:spermidine and mixed with DNA vaccine (pVAXrbd), which led to the formation of particles of combined coronavirus vaccine (CCV-RBD) that contain the DNA vaccine inside and RBD protein on the surface. CCV-RBD particles were characterized with gel filtration, electron microscopy, and biolayer interferometry. To investigate the immunogenicity of the combined vaccine and its components, mice were immunized with the DNA vaccine pVAXrbd or RBD protein as well as CCV-RBD particles. The highest antigen-specific IgG and neutralizing activity were induced by CCV-RBD, and the level of antibodies induced by DNA or RBD alone was significantly lower. The cellular immune response was detected only in the case of DNA or CCV-RBD vaccination. These results demonstrate that a combination of DNA vaccine and RBD protein in one construct synergistically increases the humoral response to RBD protein in mice.


Subject(s)
COVID-19 Vaccines/chemistry , COVID-19 Vaccines/pharmacology , Immunity, Humoral/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Animals , Binding Sites , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Dextrans/chemistry , Female , HEK293 Cells , Humans , Mice, Inbred BALB C , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spermidine/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, DNA/pharmacology , Vero Cells
2.
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.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1650946

ABSTRACT

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.


Subject(s)
Amino Alcohols/pharmacology , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/pharmacology , Phenyl Ethers/pharmacology , Receptors, Virus/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Sulfhydryl Compounds/pharmacology , Allosteric Regulation , Amino Alcohols/chemistry , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Line , Disulfides/antagonists & inhibitors , Disulfides/chemistry , Disulfides/metabolism , Dose-Response Relationship, Drug , Humans , Molecular Docking Simulation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Oxidation-Reduction , Phenyl Ethers/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Sulfhydryl Compounds/chemistry
4.
Int J Mol Sci ; 23(2)2022 Jan 12.
Article in English | MEDLINE | ID: covidwho-1623730

ABSTRACT

Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta's spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spike's molecular interactions with syndecan-4 also involve syndecan-4's cell-binding domain that mediates cell-to-cell adhesion. Regardless of the complexity of these interactions, exogenously added heparin blocks Delta's cellular entry as efficiently as syndecan-4 knockdown. Therefore, a profound understanding of the molecular mechanisms underlying Delta infections enables the development of molecularly targeted yet simple strategies to reduce the Delta variant's spread.


Subject(s)
COVID-19/transmission , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Syndecan-4/metabolism , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Line , Heparan Sulfate Proteoglycans/antagonists & inhibitors , Heparan Sulfate Proteoglycans/metabolism , Humans , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Syndecan-4/genetics , Virus Internalization
5.
Microbiol Spectr ; 10(1): e0052221, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622001

ABSTRACT

Heme-containing peroxidases are widely distributed in the animal and plant kingdoms and play an important role in host defense by generating potent oxidants. Myeloperoxidase (MPO), the prototype of heme-containing peroxidases, exists in neutrophils and monocytes. MPO has a broad spectrum of microbial killing. The difficulty of producing MPO at a large scale hinders its study and utilization. This study aimed to overexpress recombinant human MPO and characterize its microbicidal activities in vitro and in vivo. A human HEK293 cell line stably expressing recombinant MPO (rMPO) was established as a component of this study. rMPO was overexpressed and purified for studies on its biochemical and enzymatic properties, as well as its microbicidal activities. In this study, rMPO was secreted into culture medium as a monomer. rMPO revealed enzymatic activity similar to that of native MPO. rMPO, like native MPO, was capable of killing a broad spectrum of microorganisms, including Gram-negative and -positive bacteria and fungi, at low nM levels. Interestingly, rMPO could kill antibiotic-resistant bacteria, making it very useful for treatment of nosocomial infections and mixed infections. The administration of rMPO significantly reduced the morbidity and mortality of murine lung infections induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. In animal safety tests, the administration of 100 nM rMPO via tail vein did not result in any sign of toxic effects. Taken together, the data suggest that rMPO purified from a stably expressing human cell line is a new class of antimicrobial agents with the ability to kill a broad spectrum of pathogens, including bacteria and fungi with or without drug resistance. IMPORTANCE Over the past 2 decades, more than 20 new infectious diseases have emerged. Unfortunately, novel antimicrobial therapeutics are discovered at much lower rates. Infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness, greater risk of death, and high health care costs. Currently, this is best seen with the lack of a cure for coronavirus disease 2019 (COVID-19). To combat such untreatable microorganisms, there is an urgent need to discover new classes of antimicrobial agents. Myeloperoxidase (MPO) plays an important role in host defense. The difficulty of producing MPO on a large scale hinders its study and utilization. We have produced recombinant MPO at a large scale and have characterized its antimicrobial activities. Most importantly, recombinant MPO significantly reduced the morbidity and mortality of murine pneumonia induced by Pseudomonas aeruginosa or methicillin-resistant Staphylococcus aureus. Our data suggest that recombinant MPO from human cells is a new class of antimicrobials with a broad spectrum of activity.


Subject(s)
Anti-Infective Agents/pharmacology , Peroxidase/pharmacology , Acute Disease , Animals , Anti-Infective Agents/classification , Anti-Infective Agents/therapeutic use , Anti-Infective Agents/toxicity , Candida albicans/drug effects , Drug Resistance, Bacterial , Escherichia coli/drug effects , Female , HEK293 Cells , Humans , Hydrogen Peroxide/toxicity , Male , Methicillin-Resistant Staphylococcus aureus/drug effects , Mice , Mice, Inbred C57BL , Peroxidase/genetics , Peroxidase/therapeutic use , Peroxidase/toxicity , Pneumonia, Bacterial/drug therapy , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Recombinant Proteins/toxicity , Staphylococcal Infections/drug therapy , Staphylococcus aureus/drug effects
6.
Int J Biol Macromol ; 197: 68-76, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1587673

ABSTRACT

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.


Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Amyloidosis/metabolism , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Protein Domains/physiology , Amyloidosis/genetics , Coronavirus 3C Proteases/genetics , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Kinetics , Models, Molecular , Molecular Dynamics Simulation , Mutation , Polymerization , Protein Conformation, alpha-Helical , Protein Domains/genetics , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Temperature
7.
J Med Virol ; 94(4): 1633-1640, 2022 04.
Article in English | MEDLINE | ID: covidwho-1568204

ABSTRACT

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.


Subject(s)
Antibodies, Monoclonal/immunology , COVID-19 Serological Testing/instrumentation , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/isolation & purification , Animals , COVID-19/blood , COVID-19/diagnosis , COVID-19 Serological Testing/methods , Coronavirus Nucleocapsid Proteins/blood , Coronavirus Nucleocapsid Proteins/genetics , Humans , Immunoassay , Mice , Mutation , Phosphoproteins/blood , Phosphoproteins/genetics , Phosphoproteins/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity
8.
Nat Commun ; 12(1): 3172, 2021 05 26.
Article in English | MEDLINE | ID: covidwho-1550281

ABSTRACT

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.


Subject(s)
Nerve Tissue Proteins/ultrastructure , Neuropilin-1/ultrastructure , Receptors, Cell Surface/ultrastructure , Semaphorin-3A/ultrastructure , Animals , COS Cells , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/metabolism , Neuropilin-1/genetics , Neuropilin-1/isolation & purification , Neuropilin-1/metabolism , Protein Binding/genetics , Protein Domains/genetics , Protein Multimerization/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Receptors, Cell Surface/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Semaphorin-3A/genetics , Semaphorin-3A/isolation & purification , Semaphorin-3A/metabolism
9.
Biomolecules ; 11(12)2021 12 02.
Article in English | MEDLINE | ID: covidwho-1551563

ABSTRACT

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight the current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , Cell Line , Escherichia coli/genetics , Gene Expression , HEK293 Cells , Humans , Insecta/cytology , Protein Binding , Protein Denaturation , Protein Domains , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
10.
Biochem Biophys Res Commun ; 587: 69-77, 2022 01 08.
Article in English | MEDLINE | ID: covidwho-1540389

ABSTRACT

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is known to associate with AP180. An AP180 N-terminal homology domain (1-289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38-K39-K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages.


Subject(s)
Clathrin/metabolism , Docosahexaenoic Acids/metabolism , Monomeric Clathrin Assembly Proteins/metabolism , Phosphatidic Acids/metabolism , Animals , Binding Sites , Brain/metabolism , COVID-19/metabolism , COVID-19/virology , Cell Line , Clathrin/chemistry , Docosahexaenoic Acids/chemistry , Endocytosis/physiology , Host Microbial Interactions/physiology , Humans , Mice , Monomeric Clathrin Assembly Proteins/chemistry , Monomeric Clathrin Assembly Proteins/genetics , Phosphatidic Acids/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/physiology , Virus Internalization
11.
Biotechnol Lett ; 44(1): 45-57, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1536319

ABSTRACT

After its emergence in late 2019 SARS-CoV-2 was declared a pandemic by the World Health Organization on 11 March 2020 and has claimed more than 2.8 million lives. There has been a massive global effort to develop vaccines against SARS-CoV-2 and the rapid and low cost production of large quantities of vaccine is urgently needed to ensure adequate supply to both developed and developing countries. Virus-like particles (VLPs) are composed of viral antigens that self-assemble into structures that mimic the structure of native viruses but lack the viral genome. Thus they are not only a safer alternative to attenuated or inactivated vaccines but are also able to induce potent cellular and humoral immune responses and can be manufactured recombinantly in expression systems that do not require viral replication. VLPs have successfully been produced in bacteria, yeast, insect and mammalian cell cultures, each production platform with its own advantages and limitations. Plants offer a number of advantages in one production platform, including proper eukaryotic protein modification and assembly, increased safety, low cost, high scalability as well as rapid production speed, a critical factor needed to control outbreaks of potential pandemics. Plant-based VLP-based viral vaccines currently in clinical trials include, amongst others, Hepatitis B virus, Influenza virus and SARS-CoV-2 vaccines. Here we discuss the importance of plants as a next generation expression system for the fast, scalable and low cost production of VLP-based vaccines.


Subject(s)
COVID-19 Vaccines/biosynthesis , Plants, Genetically Modified/metabolism , SARS-CoV-2/immunology , Vaccines, Virus-Like Particle/biosynthesis , Antigens, Viral/genetics , Antigens, Viral/metabolism , COVID-19 Vaccines/economics , COVID-19 Vaccines/genetics , Gene Expression , Plants, Genetically Modified/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vaccines, Virus-Like Particle/economics , Vaccines, Virus-Like Particle/genetics , Viral Vaccines/biosynthesis , Viral Vaccines/genetics
12.
Microbiol Spectr ; 9(3): e0067221, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1532977

ABSTRACT

Here, we aimed to investigate the diagnostic value of a serological assay using the nucleocapsid protein developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and evaluated its performance using three commercial enzyme-linked immunosorbent assays (ELISAs), namely, Standard E 2019 novel coronavirus disease (COVID-19) total antibody (Ab) ELISA (SD Biosensor), and EDI novel coronavirus COVID-19 IgG and IgM ELISA. A recombinant nucleocapsid protein (rNP) was expressed from plants and Escherichia coli for the detection of serum total Ab. We prospectively collected 141 serum samples from 32 patients with reverse transcription-PCR (RT-PCR)-confirmed COVID-19 and determined the sensitivity and dynamics of their total Ab response. Specificity was evaluated using 158 prepandemic samples. To validate the assays, we evaluated the performance using two different cutoff values. The sensitivity and specificity for each assay were as follows: 92.91% and 94.30% (plant-rNP), 83.69% and 98.73% (SD Biosensor), 75.89% and 98.10% (E. coli-rNP), 76.47% and 100% (EDI-IgG), and 80.39% and 80% (EDI-IgM). The plant-based rNP showed the highest sensitivity and area under the receiver operating characteristic (ROC) curve (0.980) among all the assays (P < 0.05). The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs. After 2 weeks of PSO, the seroconversion rates were >80% and 100% for EDI-IgM and EDI-IgG ELISA, respectively. Seroconversion occurred earlier with rNP plant-based ELISA (5 days PSO) compared with E. coli-based (7 days PSO) and SD Biosensor (8 days PSO) ELISA. We determined that rNP produced in plants enables the robust detection of SARS-CoV-2 total Abs. The assay can be used for serosurvey and complementary diagnosis of COVID-19. IMPORTANCE At present, the principal diagnostic methods for COVID-19 comprise the identification of viral nucleic acid by genetic approaches, including PCR-based techniques or next-generation sequencing. However, there is an urgent need for validated serological assays which are crucial for the understanding of immune responses against SARS-CoV-2. In this study, a highly sensitive and specific serological antibody assay was developed for the detection of SARS-CoV-2 with an overall accuracy of 93.56% using a recombinant nucleoprotein expressed from plants.


Subject(s)
Antibodies, Viral/blood , COVID-19 Testing/methods , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Nucleocapsid Proteins/immunology , Plant Proteins/immunology , Escherichia coli/genetics , Humans , Immunoglobulin G , Immunoglobulin M , Nucleocapsid , Plant Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Seroconversion , Tobacco/genetics
13.
PLoS One ; 16(11): e0257089, 2021.
Article in English | MEDLINE | ID: covidwho-1523422

ABSTRACT

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.


Subject(s)
Chlamydomonas reinhardtii , Protein Interaction Domains and Motifs , Recombinant Proteins , Spike Glycoprotein, Coronavirus , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Cloning, Molecular , Humans , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification
14.
Glycobiology ; 32(1): 60-72, 2022 02 26.
Article in English | MEDLINE | ID: covidwho-1501077

ABSTRACT

Extensive glycosylation of the spike protein of severe acute respiratory syndrome coronavirus 2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the coronavirus disease of 2019 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain (NTD) that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific NTD site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.


Subject(s)
COVID-19/epidemiology , Glycopeptides/chemistry , Mutation , Polysaccharides/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/transmission , COVID-19/virology , Carbohydrate Sequence , Datasets as Topic , Glycopeptides/genetics , Glycopeptides/metabolism , Glycosylation , HEK293 Cells , Humans , Mass Spectrometry , Peptide Mapping , Polysaccharides/metabolism , Protein Binding , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
15.
Nat Commun ; 12(1): 6103, 2021 10 20.
Article in English | MEDLINE | ID: covidwho-1475296

ABSTRACT

Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/isolation & purification , Angiotensin-Converting Enzyme 2/ultrastructure , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , SARS-CoV-2/genetics , Sf9 Cells , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/ultrastructure , Spodoptera , Surface Plasmon Resonance , Virus Attachment , Virus Internalization
16.
Biochemistry (Mosc) ; 86(10): 1275-1287, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1476404

ABSTRACT

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.


Subject(s)
COVID-19 Vaccines , COVID-19 , Epitopes , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , COVID-19 Vaccines/isolation & purification , COVID-19 Vaccines/pharmacology , Epitopes/genetics , Epitopes/immunology , Epitopes/isolation & purification , Epitopes/pharmacology , Female , Humans , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification , Spike Glycoprotein, Coronavirus/pharmacology
17.
Molecules ; 26(19)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1463766

ABSTRACT

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-ß-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.


Subject(s)
Bacterial Proteins/metabolism , Chitin/metabolism , Chitinases/metabolism , Clostridium/metabolism , Bacterial Proteins/genetics , Catalytic Domain , Chitinases/chemistry , Chitinases/genetics , Clostridium/growth & development , Clostridium/isolation & purification , Gastrointestinal Microbiome , Humans , Hydrogen-Ion Concentration , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
18.
Mol Med ; 27(1): 120, 2021 09 26.
Article in English | MEDLINE | ID: covidwho-1440900

ABSTRACT

BACKGROUND: Since fall 2019, SARS-CoV-2 spread world-wide, causing a major pandemic with estimated ~ 220 million subjects affected as of September 2021. Severe COVID-19 is associated with multiple organ failure, particularly of lung and kidney, but also grave neuropsychiatric manifestations. Overall mortality reaches > 2%. Vaccine development has thrived in thus far unreached dimensions and will be one prerequisite to terminate the pandemic. Despite intensive research, however, few treatment options for modifying COVID-19 course/outcome have emerged since the pandemic outbreak. Additionally, the substantial threat of serious downstream sequelae, called 'long COVID' and 'neuroCOVID', becomes increasingly evident. Among candidates that were suggested but did not yet receive appropriate funding for clinical trials is recombinant human erythropoietin. Based on accumulating experimental and clinical evidence, erythropoietin is expected to (1) improve respiration/organ function, (2) counteract overshooting inflammation, (3) act sustainably neuroprotective/neuroregenerative. Recent counterintuitive findings of decreased serum erythropoietin levels in severe COVID-19 not only support a relative deficiency of erythropoietin in this condition, which can be therapeutically addressed, but also made us coin the term 'hypoxia paradox'. As we review here, this paradox is likely due to uncoupling of physiological hypoxia signaling circuits, mediated by detrimental gene products of SARS-CoV-2 or unfavorable host responses, including microRNAs or dysfunctional mitochondria. Substitution of erythropoietin might overcome this 'hypoxia paradox' caused by deranged signaling and improve survival/functional status of COVID-19 patients and their long-term outcome. As supporting hints, embedded in this review, we present 4 male patients with severe COVID-19 and unfavorable prognosis, including predicted high lethality, who all profoundly improved upon treatment which included erythropoietin analogues. SHORT CONCLUSION: Substitution of EPO may-among other beneficial EPO effects in severe COVID-19-circumvent downstream consequences of the 'hypoxia paradox'. A double-blind, placebo-controlled, randomized clinical trial for proof-of-concept is warranted.


Subject(s)
COVID-19/complications , COVID-19/drug therapy , Erythropoietin/genetics , Hypoxia/drug therapy , Lung/drug effects , COVID-19/genetics , COVID-19/pathology , COVID-19/virology , Erythropoietin/analogs & derivatives , Erythropoietin/therapeutic use , Humans , Hypoxia/genetics , Hypoxia/pathology , Hypoxia/virology , Lung/pathology , Lung/virology , Pandemics , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , SARS-CoV-2/drug effects
19.
Int J Biol Macromol ; 190: 409-416, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1412931

ABSTRACT

Coronavirus Disease 2019 (COVID-19) caused by a novel betacoronavirus SARS-CoV-2 has been an ongoing global pandemic. Several vaccines have been developed to control the COVID-19, but the potential effectiveness of the mucosal vaccine remains to be documented. In this study, we constructed a recombinant L. plantarum LP18:RBD expressing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein via the surface anchoring route. The amount of the RBD protein was maximally expressed under the culture condition with 200 ng/mL of inducer at 33 °C for 6 h. Further, we evaluated the immune response in mice via the intranasal administration of LP18:RBD. The results showed that the LP18:RBD significantly elicited RBD-specific mucosal IgA antibodies in respiratory tract and intestinal tract. The percentages of CD3 + CD4+ T cells in spleens of mice administrated with the LP18:RBD were also significantly increased. This indicated that LP18:RBD could induce a humoral immune response at the mucosa, and it could be used as a mucosal vaccine candidate against the SARS-CoV-2 infection. We provided the first experimental evidence that the recombinant L. plantarum LP18:RBD could initiate immune response in vivo, which implies that the mucosal immunization using recombinant LAB system could be a promising vaccination strategy to prevent the COVID-19 pandemic.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunity, Mucosal , Immunoglobulin A/immunology , Lactobacillus plantarum , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Administration, Intranasal , Animals , COVID-19/genetics , COVID-19/prevention & control , Gene Expression , Lactobacillus plantarum/genetics , Lactobacillus plantarum/immunology , Mice , Mice, Inbred BALB C , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
20.
PLoS One ; 16(9): e0257351, 2021.
Article in English | MEDLINE | ID: covidwho-1406752

ABSTRACT

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase-quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98-0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24-0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92-0.99), specificity (range 0.93-0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87-0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus.


Subject(s)
Antibodies, Viral/blood , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Panama/epidemiology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics
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