Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 22
Filter
1.
Front Public Health ; 10: 847384, 2022.
Article in English | MEDLINE | ID: covidwho-1792872

ABSTRACT

Background: Immunity and clinical protection induced by mRNA vaccines against SARS-CoV-2 have been shown to decline overtime. To gather information on the immunity profile deemed sufficient in protecting against hospitalization, we tested IgG levels, interferon-gamma (IFN-γ) secretion, and neutralizing antibodies 180 days (d180) after the second shot of BNT162b vaccine, in HW. Methods: A total of 392 subjects were enrolled. All received BioNTech/Pfizer from February 2020 to April 2021. The vaccine-specific humoral response was quantitatively determined by testing for IgG anti-S1 domain of SARS-CoV-spike protein. Live virus microneutralization (MN) was evaluated by an assay performing incubation of serial 2-fold dilution of human serum samples, starting from 1:10 to 1:5120, with an equal volume of Wuhan strain and Delta VOC viral solution and assessing the presence/absence of a cytopathic effect. SARS-CoV-2-spike protein-specific T-cell response was determined by a commercial IFN-γ release assay. Results: In 352 individuals, at d180, IgG levels decreased substantially but no results below the assay's positivity threshold were observed. Overall, 22 naive (8.1%) had values above the highest threshold. Among COVID-naive, the impact of age, which was observed at earlier stages, disappeared at d180, while it remained significant for 81 who had experienced a previous infection. Following the predictive model of protection by Khoury, we transformed the neutralizing titers in IU/ml and used a 54 IU/ml threshold to identify subjects with 50% protective immunity. Overall, live virus MN showed almost all subjects with previous exposure to SARS-CoV-2 neutralized the virus as compared to 33% of naive double-dosed subjects (p < 0.0001). All previously exposed subjects had strong IFN-γ secretion (>200 mIU/ml); among 271 naive, 7 (2.58%) and 17 (6.27%) subjects did not show borderline or strong secretion, respectively. Conclusions: In naive subjects, low IgG titers are relatively long-lasting. Only a third of naive subjects maintain neutralizing responses. After specific stimulation, a very limited number of naive were unable to produce IFN-γ. The results attained in the small group of subjects with breakthrough infection suggest that simultaneous neutralizing antibody titers <20, binding antibody levels/ml <200, and IFN-γ <1,000 mIU/ml in subjects older than 58 may identify at-risk groups.


Subject(s)
COVID-19 Vaccines , COVID-19 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral , COVID-19/prevention & control , Health Personnel , Humans , Immunity, Humoral , Immunoglobulin G , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/pharmacology
2.
Curr Med Chem ; 29(4): 682-699, 2022.
Article in English | MEDLINE | ID: covidwho-1742083

ABSTRACT

COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin- converting enzyme 2 (ACE2), as well as the cleavage effect of a type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Humans , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/pharmacology , Virus Internalization
3.
PLoS One ; 17(3): e0263671, 2022.
Article in English | MEDLINE | ID: covidwho-1742001

ABSTRACT

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/µm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future.


Subject(s)
COVID-19 Vaccines , COVID-19 , Erythrocyte Membrane , Molecular Dynamics Simulation , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus , Vaccines, Virus-Like Particle , Animals , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , Erythrocyte Membrane/chemistry , Erythrocyte Membrane/immunology , Female , Liposomes , Mice , Pilot Projects , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/pharmacology , Vaccines, Virus-Like Particle/chemistry , Vaccines, Virus-Like Particle/immunology , Vaccines, Virus-Like Particle/pharmacology
4.
J Food Biochem ; 46(5): e14062, 2022 05.
Article in English | MEDLINE | ID: covidwho-1627170

ABSTRACT

Therapeutic drugs based on natural products for the treatment of SARS-CoV-2 are currently unavailable. This study was conducted to develop an anti-SARS-CoV-2 herbal medicine to face the urgent need for COVID-19 treatment. The bioactive components from ethanolic extract of Moringa oleifera fruits (MOFs) were determined by gas chromatography-mass spectroscopy (GC-MS). Molecular-docking analyses elucidated the binding effects of identified phytocomponents against SARS-CoV-2 spike glycoprotein (PDB ID: 6VYB) and human ACE2 receptor (PDB ID: 1R42) through the Glide module of Maestro software. GC-MS analysis unveiled the presence of 33 phytocomponents. Eighteen phytocomponents exhibited good binding affinity toward ACE2 receptor, and thirteen phytocomponents had a high affinity with spike glycoprotein. This finding suggests that the top 11 hits (Docking score ≥ -3.0 kcal/mol) could inhibit SARS-CoV-2 propagation. Intriguingly, most of the phytoconstituents displayed drug-likeness with no predicted toxicity. However, further studies are needed to validate their effects and mechanisms of action. PRACTICAL APPLICATIONS: Moringa oleifera (MO) also called "drumstick tree" has been used as an alternative food source to combat malnutrition and may act as an immune booster. GC-MS analysis unveiled that ethanolic extract of Moringa oleifera fruits (MOFs) possessed 33 active components of pyridine, aromatic fatty acid, oleic acid, tocopherol, methyl ester, diterpene alcohol, triterpene and fatty acid ester and their derivatives, which have various pharmacological and medicinal values. Virtual screening study of phytocomponents of MOF with human ACE2 receptor and SARS-CoV-2 spike glycoprotein exhibited good binding affinity. Based on molecular docking, the top 11 hits (Docking score ≥-3.0 kcal/mol) might serve as potential lead molecules in antiviral drug development. Intriguingly, most of the phytoconstituents displayed drug-likeness with no predicted toxicity. Thus, MOF might be used as a valuable source for antiviral drug development to combat COVID-19, an ongoing pandemic.


Subject(s)
Antiviral Agents , Moringa oleifera , Plant Extracts , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Esters/pharmacology , Fatty Acids/pharmacology , Fruit/metabolism , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Moringa oleifera/chemistry , Phytochemicals/pharmacology , Plant Extracts/pharmacology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/pharmacology
5.
Biosci Rep ; 41(12)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1592575

ABSTRACT

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Endotoxins/pharmacology , Peptide Fragments/pharmacology , Spike Glycoprotein, Coronavirus/pharmacology , Alphacoronavirus , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , CD13 Antigens/metabolism , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cricetulus , Endotoxins/toxicity , Hemolysis/drug effects , Humans , Molecular Docking Simulation , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Conformation, alpha-Helical , Sheep, Domestic , Spike Glycoprotein, Coronavirus/toxicity , Structure-Activity Relationship
6.
Biosci Rep ; 41(12)2021 12 22.
Article in English | MEDLINE | ID: covidwho-1526113

ABSTRACT

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and ß-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Endotoxins/pharmacology , Peptide Fragments/pharmacology , Spike Glycoprotein, Coronavirus/pharmacology , Alphacoronavirus , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/toxicity , CD13 Antigens/metabolism , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cricetulus , Endotoxins/toxicity , Hemolysis/drug effects , Humans , Molecular Docking Simulation , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Conformation, alpha-Helical , Sheep, Domestic , Spike Glycoprotein, Coronavirus/toxicity , Structure-Activity Relationship
7.
Neurobiol Dis ; 161: 105561, 2021 12.
Article in English | MEDLINE | ID: covidwho-1510138

ABSTRACT

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Brain/metabolism , COVID-19/metabolism , Hypoxia-Ischemia, Brain/metabolism , Hypoxia/metabolism , Inflammation/metabolism , Pericytes/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Actins/metabolism , Angiotensin-Converting Enzyme 2/drug effects , Angiotensin-Converting Enzyme 2/genetics , Animals , Brain/blood supply , COVID-19/physiopathology , Calcium Signaling , Collagen Type I/metabolism , Fibronectins/metabolism , Humans , Hypoxia-Ischemia, Brain/physiopathology , Lipid Peroxidation/drug effects , Lipid Peroxidation/genetics , Macrophage Migration-Inhibitory Factors/drug effects , Macrophage Migration-Inhibitory Factors/metabolism , Mice , Mice, Transgenic , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Myofibroblasts , NF-kappa B/drug effects , NF-kappa B/metabolism , Nasal Mucosa , Nitrosative Stress , Oxidative Stress , Pericytes/cytology , Pericytes/drug effects , Phenotype , Receptor, Notch3/metabolism , Receptors, Coronavirus/drug effects , Receptors, Coronavirus/genetics , Receptors, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/pharmacology
8.
Int J Biol Macromol ; 193(Pt B): 1885-1897, 2021 Dec 15.
Article in English | MEDLINE | ID: covidwho-1509845

ABSTRACT

The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.


Subject(s)
Adjuvants, Immunologic , Antibodies, Viral/immunology , Broadly Neutralizing Antibodies/immunology , COVID-19 Vaccines , Immunogenicity, Vaccine , Nanoparticles , Oligodeoxyribonucleotides , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus , Th1 Cells/immunology , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/pharmacology , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/pharmacology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/pharmacology
9.
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
10.
Protein Expr Purif ; 190: 106003, 2022 02.
Article in English | MEDLINE | ID: covidwho-1474960

ABSTRACT

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.


Subject(s)
COVID-19 Vaccines , Gene Expression , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , COVID-19 Vaccines/pharmacology , Humans , Mice , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/pharmacology
11.
Molecules ; 26(20)2021 Oct 14.
Article in English | MEDLINE | ID: covidwho-1470935

ABSTRACT

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.


Subject(s)
Asparagus Plant/chemistry , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Macrophages/drug effects , Plant Extracts/pharmacology , Signal Transduction/drug effects , Animals , Asparagus Plant/metabolism , Butadienes/pharmacology , Cell Survival/drug effects , Interleukin-1beta/genetics , Interleukin-6/genetics , Macrophages/cytology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Nitriles/pharmacology , Phosphorylation/drug effects , Plant Extracts/chemistry , Plant Stems/chemistry , Plant Stems/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Spike Glycoprotein, Coronavirus/pharmacology , Toll-Like Receptor 4/metabolism , Transcription, Genetic/drug effects
12.
Int J Biol Sci ; 17(14): 3786-3794, 2021.
Article in English | MEDLINE | ID: covidwho-1417292

ABSTRACT

COVID-19, caused by a novel coronavirus, SARS-CoV-2, poses a serious global threat. It was first reported in 2019 in China and has now dramatically spread across the world. It is crucial to develop therapeutics to mitigate severe disease and viral spread. The receptor-binding domains (RBDs) in the spike protein of SARS-CoV and MERS-CoV have shown anti-viral activity in previous reports suggesting that this domain has high potential for development as therapeutics. To evaluate the potential antiviral activity of recombinant SARS-CoV-2 RBD proteins, we determined the RBD residues of SARS-CoV-2 using a homology search with RBD of SARS-CoV. For efficient expression and purification, the signal peptide of spike protein was identified and used to generate constructs expressing recombinant RBD proteins. Highly purified RBD protein fused with the Fc domain of human IgG showed potent anti-viral efficacy, which was better than that of a protein fused with a histidine tag. Intranasally pre-administrated RBD protein also inhibited the attachment of SARS-COV-2 to mouse lungs. These findings indicate that RBD protein could be used for the prevention and treatment of SARS-CoV-2 infection.


Subject(s)
COVID-19/drug therapy , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/therapeutic use , Virus Attachment/drug effects , Administration, Intranasal , Amino Acid Sequence , Animals , Binding Sites , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Mice, Inbred C57BL , Microbial Sensitivity Tests , Protein Domains , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/pharmacology , Vero Cells
13.
Int J Mol Sci ; 22(14)2021 Jul 14.
Article in English | MEDLINE | ID: covidwho-1314666

ABSTRACT

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Cytokines/metabolism , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Models, Biological , Spike Glycoprotein, Coronavirus/pharmacology , Healthy Volunteers , Humans , In Vitro Techniques , Leukocytes, Mononuclear/drug effects
15.
Curr Med Chem ; 29(4): 682-699, 2022.
Article in English | MEDLINE | ID: covidwho-1197459

ABSTRACT

COVID-19 is an infectious disease caused by SARS-CoV-2. The life cycle of SARS-CoV-2 includes the entry into the target cells, replicase translation, replicating and transcribing genomes, translating structural proteins, assembling and releasing new virions. Entering host cells is a crucial stage in the early life cycle of the virus, and blocking this stage can effectively prevent virus infection. SARS enters the target cells mediated by the interaction between the viral S protein and the target cell surface receptor angiotensin- converting enzyme 2 (ACE2), as well as the cleavage effect of a type-II transmembrane serine protease (TMPRSS2) on the S protein. Therefore, the ACE2 receptor and TMPRSS2 are important targets for SARS-CoV-2 entry inhibitors. Herein, we provide a concise report/information on drugs with potential therapeutic value targeting virus-ACE2 or virus-TMPRSS2 interactions to provide a reference for the design and discovery of potential entry inhibitors against SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , COVID-19/drug therapy , Humans , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/pharmacology , Virus Internalization
16.
Front Immunol ; 12: 637982, 2021.
Article in English | MEDLINE | ID: covidwho-1156123

ABSTRACT

A novel betacoronavirus (SARS-CoV-2) that causes severe pneumonia emerged through zoonosis in late 2019. The disease, referred to as COVID-19, has an alarming mortality rate and it is having a devastating effect on the global economy and public health systems. A safe, effective vaccine is urgently needed to halt this pandemic. In this study, immunogenicity of the receptor binding domain (RBD) of spike (S) glycoprotein was examined in mice. Animals were immunized with recombinant RBD antigen intraperitoneally using three different adjuvants (Zn-chitosan, Alhydrogel, and Adju-Phos), and antibody responses were followed for over 5 months. Results showed that potent neutralizing antibodies (nAbs) can be induced with 70% neutralization titer (NT70) of ~14,580 against live, infectious viruses. Although antigen-binding antibody titers decreased gradually over time, sufficiently protective levels of nAbs persisted (NT80 >2,430) over the 5-month observation period. Results also showed that adjuvants have profound effects on kinetics of nAb induction, total antibody titers, antibody avidity, antibody longevity, and B-cell epitopes targeted by the immune system. In conclusion, a recombinant subunit protein immunogen based on the RBD is a highly promising vaccine candidate. Continued evaluation of RBD immunogenicity using different adjuvants and vaccine regimens could further improve vaccine efficacy.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/pharmacology , COVID-19/prevention & control , Immunization , Immunogenicity, Vaccine , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/pharmacology , Adjuvants, Immunologic/pharmacology , Animals , Antibody Affinity , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Epitopes , Female , Host-Pathogen Interactions , Mice, Inbred BALB C , Protein Domains , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Vaccines, Subunit/immunology , Vaccines, Subunit/pharmacology
17.
Sci Adv ; 7(12)2021 03.
Article in English | MEDLINE | ID: covidwho-1142982

ABSTRACT

Vaccination against SARS-CoV-2 provides an effective tool to combat the COVID-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2GΔHR2 elicited twofold higher NAb titers than S2P, while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T cell immunity, thereby providing a promising vaccine candidate.


Subject(s)
COVID-19 Vaccines , COVID-19/immunology , Nanoparticles , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , HEK293 Cells , Humans , Immunogenicity, Vaccine , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Protein Domains , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/pharmacology
18.
Nanomedicine ; 34: 102372, 2021 06.
Article in English | MEDLINE | ID: covidwho-1117330

ABSTRACT

The development of vaccines is a crucial response against the COVID-19 pandemic and innovative nanovaccines could increase the potential to address this remarkable challenge. In the present study a B cell epitope (S461-493) from the spike protein of SARS-CoV-2 was selected and its immunogenicity validated in sheep. This synthetic peptide was coupled to gold nanoparticles (AuNP) functionalized with SH-PEG-NH2 via glutaraldehyde-mediated coupling to obtain the AuNP-S461-493 candidate, which showed in s.c.-immunized mice a superior immunogenicity (IgG responses) when compared to soluble S461-493; and led to increased expression of relevant cytokines in splenocyte cultures. Interestingly, the response triggered by AuNP-S461-493 was similar in magnitude to that induced using a conventional strong adjuvant (Freund's adjuvant). This study provides a platform for the development of AuNP-based nanovaccines targeting specific SARS-CoV-2 epitopes.


Subject(s)
COVID-19 Vaccines , Epitopes, B-Lymphocyte , Gold , Immunogenicity, Vaccine , Metal Nanoparticles , Peptides , Spike Glycoprotein, Coronavirus , Animals , COVID-19 Vaccines/chemical synthesis , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , COVID-19 Vaccines/pharmacology , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Epitopes, B-Lymphocyte/pharmacology , Gold/chemistry , Gold/pharmacology , HEK293 Cells , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/therapeutic use , Mice , Mice, Inbred BALB C , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Peptides/pharmacology , Sheep , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/pharmacology
19.
Biochem Biophys Res Commun ; 546: 97-102, 2021 03 26.
Article in English | MEDLINE | ID: covidwho-1062239

ABSTRACT

The SARS-CoV-2 virus causes elevated production of senescence-associated secretory phenotype (SASP) markers by macrophages. SARS-CoV-2 enters macrophages through its Spike-protein aided by cathepsin (Cat) B and L, which also mediate SASP production. Since M-CSF and IL-34 control macrophage differentiation, we investigated the age-dependent effects of the Spike-protein on SASP-related pro-inflammatory-cytokines and nuclear-senescence-regulatory-factors, and CatB, L and K, in mouse M-CSF- and IL-34-differentiated macrophages. The Spike-protein upregulated SASP expression in young and aged male M-CSF-macrophages. In contrast, only young and aged male IL-34-macrophages demonstrated significantly reduced pro-inflammatory cytokine expression in response to the Spike-protein in vitro. Furthermore, the S-protein elevated CatB expression in young male M-CSF-macrophages and young female IL-34-macrophages, whereas CatL was overexpressed in young male IL-34- and old male M-CSF-macrophages. Surprisingly, the S-protein increased CatK activity in young and aged male M-CSF-macrophages, indicating that CatK may be also involved in the COVID-19 pathology. Altogether, we demonstrated the age- and sex-dependent effects of the Spike-protein on M-CSF and IL-34-macrophages using a novel in vitro mouse model of SARS-CoV-2/COVID-19.


Subject(s)
Age Factors , Macrophages/virology , Spike Glycoprotein, Coronavirus/pharmacology , Animals , Cathepsins/metabolism , Cell Differentiation , Cellular Senescence , Cytokines/metabolism , Female , Interleukins , Macrophage Colony-Stimulating Factor , Macrophages/cytology , Male , Mice , Mice, Inbred C57BL , Recombinant Proteins/pharmacology , SARS-CoV-2 , Sex Factors
SELECTION OF CITATIONS
SEARCH DETAIL