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1.
J Biosci Bioeng ; 134(5): 432-440, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2041897

ABSTRACT

The production of recombinant proteins using insect cells has been widely used for over 30 years, which contributing to life science research and biotechnology. Insect cells exhibiting enhanced N-glycosylation and recombinant protein productivity enhance the productivity of the baculovirus-insect cell system (BICS). A new highly proliferative insect cell strain, 2g2, was established from the Mamestra brassicae pupa ovary cell strain NIAS-MB-32 (RCB0413) to address the problem of Sf-rhabdovirus and to explore the newly available possibilities in BICS as well as Sf9, such as increased protein production and recombinant baculovirus amplification. The high-growth cell strain 2g2 was examined for its recombinant protein production ability and baculovirus productivity; moreover, the activity of the produced recombinant proteins was examined using Sf9 as a benchmark. Recombinant protein productivity and virus production by BICS in 2g2 was confirmed as equivalent to that of Sf9. Furthermore, we produced the severe acute respiratory syndrome coronavirus 2 spike protein in a baculovirus-free system and compared its productivity, binding activity with human angiotensin-converting enzyme 2, and N-glycosylation. The productivity and bioactivity were found to be equal to or better than that of Sf9. Moreover, N-glycosylation analysis revealed that the glycans derived from the 2g2-produced glycoproteins were mostly of the high mannose type as Sf9. Therefore, 2g2 may have the same N-glycosylation ability as Sf9. Finally, the Sf-rhabdovirus was confirmed to be negative in 2g2. Our results demonstrated that the novel insect cell strain 2g2 can serve as a protein production tool in scientific research and industrial biotechnology.


Subject(s)
Baculoviridae , COVID-19 , Animals , Humans , Baculoviridae/genetics , Baculoviridae/metabolism , Recombinant Proteins/metabolism , Insecta , Spodoptera/metabolism
2.
Trends Biotechnol ; 40(10): 1248-1260, 2022 10.
Article in English | MEDLINE | ID: covidwho-2016093

ABSTRACT

Plant expression platforms are low-cost, scalable, safe, and environmentally friendly systems for the production of recombinant proteins and bioactive metabolites. Rice (Oryza sativa L.) endosperm is an ideal bioreactor for the production and storage of high-value active substances, including pharmaceutical proteins, oral vaccines, vitamins, and nutraceuticals such as flavonoids and carotenoids. Here, we explore the use of molecular farming from producing medicines to developing functional food crops (biofortification). We review recent progress in producing pharmaceutical proteins and bioactive substances in rice endosperm and compare this platform with other plant expression systems. We describe how rice endosperm could be modified to design metabolic pathways and express and store stable products and discuss the factors restricting the commercialization of transgenic rice products and future prospects.


Subject(s)
Endosperm , Oryza , Carotenoids , Endosperm/genetics , Endosperm/metabolism , Flavonoids , Gene Expression Regulation, Plant , Molecular Farming , Oryza/genetics , Oryza/metabolism , Pharmaceutical Preparations/metabolism , Plant Proteins , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Proteins/metabolism , Vitamins/metabolism
3.
Microb Cell Fact ; 21(1): 180, 2022 Sep 05.
Article in English | MEDLINE | ID: covidwho-2009403

ABSTRACT

BACKGROUND: Komagataella phaffii is a commonly used alternative host for manufacturing therapeutic proteins, in part because of its ability to secrete recombinant proteins into the extracellular space. Incorrect processing of secreted proteins by cells can, however, cause non-functional product-related variants, which are expensive to remove in purification and lower overall process yields. The secretion signal peptide, attached to the N-terminus of the recombinant protein, is a major determinant of the quality of the protein sequence and yield. In K. phaffii, the signal peptide from the Saccharomyces cerevisiae alpha mating factor often yields the highest secreted titer of recombinant proteins, but the quality of secreted protein can vary highly. RESULTS: We determined that an aggregated product-related variant of the SARS-CoV-2 receptor binding domain is caused by N-terminal extension from incomplete cleavage of the signal peptide. We eliminated this variant and improved secreted protein titer up to 76% by extension of the N-terminus with a short, functional peptide moiety or with the EAEA residues from the native signal peptide. We then applied this strategy to three other recombinant subunit vaccine antigens and observed consistent elimination of the same aggregated product-related variant. Finally, we demonstrated that this benefit in quality and secreted titer can be achieved with addition of a single amino acid to the N-terminus of the recombinant protein. CONCLUSIONS: Our observations suggest that steric hindrance of proteases in the Golgi that cleave the signal peptide can cause unwanted N-terminal extension and related product variants. We demonstrated that this phenomenon occurs for multiple recombinant proteins, and can be addressed by minimal modification of the N-terminus to improve steric accessibility. This strategy may enable consistent secretion of a broad range of recombinant proteins with the highly productive alpha mating factor secretion signal peptide.


Subject(s)
COVID-19 , Humans , Mating Factor , Protein Sorting Signals , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2 , Saccharomyces cerevisiae/metabolism , Saccharomycetales
4.
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
5.
Viruses ; 13(12)2021 12 17.
Article in English | MEDLINE | ID: covidwho-1702075

ABSTRACT

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


Subject(s)
Lung/metabolism , Respiratory Mucosa/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Bronchi/metabolism , Cytokines/metabolism , Gene Expression Profiling , Humans , Models, Biological , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism
6.
Biochem Biophys Res Commun ; 601: 129-136, 2022 04 23.
Article in English | MEDLINE | ID: covidwho-1699331

ABSTRACT

COVID-19, caused by SARS-CoV-2, has been spreading worldwide for more than two years and has led to immense challenges to human health. Despite the great efforts that have been made, our understanding of SARS-CoV-2 is still limited. The viral helicase, NSP13 is an important enzyme involved in SARS-CoV-2 replication and transcription. Here we highlight the important role of the stalk domain in the enzymatic activity of NSP13. Without the stalk domain, NSP13 loses its dsRNA unwinding ability due to the lack of ATPase activity. The stalk domain of NSP13 also provides a rigid connection between the ZBD and helicase domain. We found that the tight connection between the stalk and helicase is necessary for NSP13-mediated dsRNA unwinding. When a short flexible linker was inserted between the stalk and helicase domains, the helicase activity of NSP13 was impaired, although its ATPase activity remained intact. Further study demonstrated that linker insertion between the stalk and helicase domains attenuated the RNA binding ability and affected the thermal stability of NSP13. In summary, our results suggest the crucial role of the stalk domain in NSP13 enzymatic activity and provide mechanistic insight into dsRNA unwinding by SARS-CoV-2 NSP13.


Subject(s)
COVID-19/prevention & control , Methyltransferases/metabolism , RNA Helicases/metabolism , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Binding Sites/genetics , COVID-19/virology , Enzyme Stability , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Mutation , Protein Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Helicases/chemistry , RNA Helicases/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Temperature , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/genetics
7.
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
8.
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
9.
Int J Mol Sci ; 23(1)2022 Jan 05.
Article in English | MEDLINE | ID: covidwho-1613826

ABSTRACT

Nucleic acid aptamers specific to S-protein and its receptor binding domain (RBD) of SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virions are of high interest as potential inhibitors of viral infection and recognizing elements in biosensors. Development of specific therapy and biosensors is complicated by an emergence of new viral strains bearing amino acid substitutions and probable differences in glycosylation sites. Here, we studied affinity of a set of aptamers to two Wuhan-type RBD of S-protein expressed in Chinese hamster ovary cell line and Pichia pastoris that differ in glycosylation patterns. The expression system for the RBD protein has significant effects, both on values of dissociation constants and relative efficacy of the aptamer binding. We propose glycosylation of the RBD as the main force for observed differences. Moreover, affinity of a several aptamers was affected by a site of biotinylation. Thus, the robustness of modified aptamers toward new virus variants should be carefully tested.


Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Immobilized Nucleic Acids/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , Binding Sites , CHO Cells , Cricetulus , Glycosylation , Protein Binding , Protein Domains , Protein Interaction Domains and Motifs , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SARS-CoV-2 , Saccharomycetales/genetics
10.
Cell Mol Immunol ; 19(2): 222-233, 2022 02.
Article in English | MEDLINE | ID: covidwho-1607212

ABSTRACT

Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.


Subject(s)
Adjuvants, Immunologic/adverse effects , COVID-19 Vaccines/adverse effects , COVID-19/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine/immunology , Pandemics/prevention & control , Polysorbates/adverse effects , SARS-CoV-2/immunology , Squalene/adverse effects , Tobacco/metabolism , Vaccination/methods , Vaccines, Virus-Like Particle/adverse effects , alpha-Tocopherol/adverse effects , Adjuvants, Immunologic/administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Disease Models, Animal , Drug Combinations , Drug Compounding/methods , Immunity, Humoral , Macaca mulatta , Male , Polysorbates/administration & dosage , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Squalene/administration & dosage , Treatment Outcome , Vaccines, Virus-Like Particle/administration & dosage , alpha-Tocopherol/administration & dosage
11.
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
12.
Emerg Microbes Infect ; 11(1): 277-283, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1585239

ABSTRACT

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.


Subject(s)
COVID-19/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization , Virus Replication , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Chlorocebus aethiops , Chloroquine/pharmacology , Endocytosis/drug effects , Esters/pharmacology , Guanidines/pharmacology , Humans , Immune Evasion , Lung Neoplasms/pathology , Macrolides/pharmacology , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , Vero Cells , Virus Cultivation , Virus Internalization/drug effects , Whole Genome Sequencing
13.
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
14.
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
15.
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
16.
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
17.
Cell Rep ; 37(3): 109838, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1517083

ABSTRACT

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads, variants with enhanced virulence and transmissibility have emerged. Although in vitro systems allow rapid characterization, they do not fully recapitulate the dynamic interaction of virions and neutralizing antibodies in the airway. Here, we demonstrate that the N501Y variant permits respiratory infection in unmodified mice. We utilize N501Y to survey in vivo pseudovirus infection dynamics and susceptibility to reinfection with the L452R (Los Angeles), K417N + E484K (South Africa), and L452R + K417N + E484Q (India) variants. Human coronavirus disease 2019 (COVID-19)+ or vaccinated antibody isotypes, titers, variant receptor binding domain (RBD) binding, and neutralization potential are studied, revealing numerous significant correlations. Immune escape of the K417N + E484K variant is observed because infection can be appreciated in the nasopharynx, but not lungs, of mice transferred with low-antibody-tier plasma. Conversely, near-complete protection is observed in animals receiving high-antibody-tier plasma, a phenomenon that can only be appreciated in vivo.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/therapy , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Cell Line , Cricetinae , Genetic Variation , HEK293 Cells , Humans , Immune System , Immunization, Passive/methods , In Vitro Techniques , Mice , Mutation , Nasopharynx/virology , Protein Binding , Recombinant Proteins/metabolism , Spike Glycoprotein, Coronavirus/genetics
18.
Toxicol Appl Pharmacol ; 434: 115796, 2022 01 01.
Article in English | MEDLINE | ID: covidwho-1510333

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required the urgent development of new therapies, among which passive immunotherapy is contemplated. CoviFab (INM005) is a RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies. We investigate their preclinical security and biodistribution by in vivo and ex vivo NIR imaging after intravenous administration of a dose of 4 mg/kg at time 0 and 48 h. Images were taken at 1, 12, 24, 36, 48, 49, 60, 72, 84, 96, 108, 120, 132 and 144 h after the first intravenous injection. At 96 and 144 h, mice were sacrificed for haematology, serum chemistry, clinical pathology, histopathology and ex vivo imaging. The biodistribution profile was similar in all organs studied, with the highest fluorescence at 1 h after each injection, gradually decreasing after that each one and until the end of the study (144 h). The toxicology study revealed no significant changes in the haematology and serum chemistry parameters. Further, there were no changes in the gross and histological examination of organs. Nonclinical data of the current study confirm that CoviFab is safe, without observable adverse effects in mice. Furthermore, we confirm that bioimaging studies are a useful approach in preclinical trials to determine biodistribution.


Subject(s)
Antibodies, Viral/metabolism , COVID-19/drug therapy , Receptors, Immunologic/metabolism , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , Administration, Intravenous , Animals , Antibodies, Viral/administration & dosage , Antibodies, Viral/adverse effects , COVID-19/metabolism , COVID-19/prevention & control , HEK293 Cells , Horses , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Receptors, Immunologic/administration & dosage , Recombinant Proteins/administration & dosage , Recombinant Proteins/adverse effects , SARS-CoV-2/drug effects , Spectroscopy, Near-Infrared/methods , Tissue Distribution/drug effects , Tissue Distribution/physiology
19.
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
20.
J Mol Biol ; 434(2): 167332, 2022 01 30.
Article in English | MEDLINE | ID: covidwho-1492301

ABSTRACT

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Mutation, Missense/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Viral/blood , Binding Sites/genetics , COVID-19/virology , Glycosylation , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Mannose/metabolism , Mutation, Missense/genetics , Oligosaccharides/metabolism , Polysaccharides/metabolism , Proline/genetics , Proline/immunology , Proline/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
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