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1.
Antiviral Res ; 200: 105290, 2022 04.
Article in English | MEDLINE | ID: covidwho-1734185

ABSTRACT

Neutralizing antibodies (NAbs) are believed to be promising prophylactic and therapeutic treatment against the coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we reported two mouse monoclonal antibodies 7 Eb-4G and 1Ba-3H that specifically recognized the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein without exhibiting cross-reactivity with the S proteins of SARS-CoV and MERS-CoV. The binding epitopes of 7 Eb-4G and 1Ba-3H were respectively located in the regions of residues 457-476 and 477-496 in the S protein. Only 1Ba-3H exhibited the neutralizing activity for preventing the pseudotyped lentivirus from binding to the angiotensin-converting enzyme 2 (ACE2)-transfected HEK293T cells. The competitive ELISA further showed that 1Ba-3H interfered with the binding between RBD and ACE2. Epitope mapping experiments demonstrated that a single alanine replacement at residues 480, 482, 484, 485, and 488-491 in the RBD abrogated 1Ba-3H binding. 1Ba-3H exhibited the neutralizing activity against the wild-type, Alpha, Delta, and Epsilon variants of SARS-CoV-2, but lost the neutralizing activity against Gamma variant in the plaque reduction assay. On the contrary, 1Ba-3H enhanced the cellular infection of Gamma variant in a dose-dependent manner. Our findings suggest that the antibody-dependent enhancement of infection mediated by the RBD-specific antibody for different SARS-CoV-2 variants must be considered while developing the NAb.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Monoclonal , Epitopes , HEK293 Cells , Humans , Mice , Spike Glycoprotein, Coronavirus
2.
Sci Transl Med ; 14(639): eabm0899, 2022 04 06.
Article in English | MEDLINE | ID: covidwho-1714341

ABSTRACT

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.


Subject(s)
COVID-19 Vaccines , Polysaccharides , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines/immunology , COVID-19 Vaccines/metabolism , Humans , Mice , Models, Animal , SARS-CoV-2 , Vaccination
3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-308052

ABSTRACT

A metal nanoparticle composite TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO 2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 can inhibit six major clades of SARS-CoV-2 with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to ACE2 receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and Tamiflu-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.

4.
EMBO Mol Med ; 14(4): e15298, 2022 04 07.
Article in English | MEDLINE | ID: covidwho-1675333

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants has altered the trajectory of the COVID-19 pandemic and raised some uncertainty on the long-term efficiency of vaccine strategy. The development of new therapeutics against a wide range of SARS-CoV-2 variants is imperative. We, here, have designed an inhalable siRNA, C6G25S, which covers 99.8% of current SARS-CoV-2 variants and is capable of inhibiting dominant strains, including Alpha, Delta, Gamma, and Epsilon, at picomolar ranges of IC50 in vitro. Moreover, C6G25S could completely inhibit the production of infectious virions in lungs by prophylactic treatment, and decrease 96.2% of virions by cotreatment in K18-hACE2-transgenic mice, accompanied by a significant prevention of virus-associated extensive pulmonary alveolar damage, vascular thrombi, and immune cell infiltrations. Our data suggest that C6G25S provides an alternative and effective approach to combating the COVID-19 pandemic.


Subject(s)
COVID-19 , Animals , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Pandemics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics
5.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-294970

ABSTRACT

A major challenge to end the pandemic caused by SARS-CoV-2 is to develop a broadly protective vaccine. As the key immunogen, the spike protein is frequently mutated with conserved epitopes shielded by glycans. Here, we reveal that spike glycosylation has site-differential effects on viral infectivity and lung epithelial cells generate spike with more infective glycoforms. Compared to the fully glycosylated spike, immunization of spike protein with N-glycans trimmed to the monoglycosylated state (S mg ) elicits stronger immune responses and better protection for hACE2 transgenic mice against variants of concern. In addition, a broadly neutralizing monoclonal antibody was identified from the S mg immunized mice, demonstrating that removal of glycan shields to better expose the conserved sequences is an effective and simple approach to broad-spectrum vaccine development. One-Sentence Summary Removing glycan shields to expose conserved epitopes is an effective approach to develop a broad-spectrum SARS-CoV-2 vaccine.

6.
Front Pharmacol ; 12: 706901, 2021.
Article in English | MEDLINE | ID: covidwho-1394795

ABSTRACT

Remdesivir, a prodrug targeting RNA-dependent-RNA-polymerase, and cyclosporine, a calcineurin inhibitor, individually exerted inhibitory activity against human coronavirus OC43 (HCoV-OC43) in HCT-8 and MRC-5 cells at EC50 values of 96 ± 34 ∼ 85 ± 23 nM and 2,920 ± 364 ∼ 4,419 ± 490 nM, respectively. When combined, these two drugs synergistically inhibited HCoV-OC43 in both HCT-8 and MRC-5 cells assayed by immunofluorescence assay (IFA). Remdesivir and cyclosporine also separately reduced IL-6 production induced by HCoV-OC43 in human lung fibroblasts MRC-5 cells with EC50 values of 224 ± 53 nM and 1,292 ± 352 nM, respectively; and synergistically reduced it when combined. Similar trends were observed for SARS-CoV-2, which were 1) separately inhibited by remdesivir and cyclosporine with respective EC50 values of 3,962 ± 303 nM and 7,213 ± 143 nM by IFA, and 291 ± 91 nM and 6,767 ± 1,827 nM by a plaque-formation assay; and 2) synergistically inhibited by their combination, again by IFA and plaque-formation assay. Collectively, these results suggest that the combination of remdesivir and cyclosporine merits further study as a possible treatment for COVID-19 complexed with a cytokine storm.

7.
mBio ; 12(4): e0058721, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1327613

ABSTRACT

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.


Subject(s)
COVID-19/transmission , Furin/metabolism , Giant Cells/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Substitution/genetics , Animals , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Furin/antagonists & inhibitors , Genetic Fitness/genetics , Genome, Viral/genetics , HEK293 Cells , Humans , SARS-CoV-2/isolation & purification , Vero Cells , Viral Load/genetics , Virus Replication/genetics
8.
Sci Rep ; 11(1): 8692, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1199310

ABSTRACT

A metal nanoparticle composite, namely TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 inhibited six major clades of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to the angiotensin-converting enzyme 2 (ACE2) receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and oseltamivir-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.


Subject(s)
Gold/pharmacology , Influenza A Virus, H1N1 Subtype/physiology , Influenza A Virus, H5N1 Subtype/physiology , SARS-CoV-2/physiology , Silver/pharmacology , Zinc Oxide/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Drug Resistance, Viral/drug effects , Food Additives/pharmacology , Gold/chemistry , HEK293 Cells , Humans , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/drug effects , Metal Nanoparticles/chemistry , Nanocomposites/chemistry , Oseltamivir/pharmacology , Particle Size , Protein Binding/drug effects , SARS-CoV-2/drug effects , Silver/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Zinc Oxide/chemistry
9.
Antimicrob Agents Chemother ; 65(4)2021 03 18.
Article in English | MEDLINE | ID: covidwho-1142997

ABSTRACT

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 µM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 µM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Peptide Hydrolases/metabolism , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Humans , Kinetics , SARS-CoV-2/metabolism , Substrate Specificity , Vero Cells
10.
Front Pharmacol ; 11: 606097, 2020.
Article in English | MEDLINE | ID: covidwho-1004688

ABSTRACT

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7-8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2-3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6-7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5-14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

11.
Heliyon ; 6(12): e05646, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-967129

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent for the outbreak of coronavirus disease 2019 (COVID-19). This global pandemic is now calling for efforts to develop more effective COVID-19 therapies. Here we use a host-directed approach, which focuses on cellular responses to diverse small-molecule treatments, to identify potentially effective drugs for COVID-19. This framework looks at the ability of compounds to elicit a similar transcriptional response to IFN-ß, a type I interferon that fails to be induced at notable levels in response to SARS-CoV-2 infection. By correlating the perturbation profiles of ~3,000 small molecules with a high-quality signature of IFN-ß-responsive genes in primary normal human bronchial epithelial cells, our analysis revealed four candidate COVID-19 compounds, namely homoharringtonine, narciclasine, anisomycin, and emetine. We experimentally confirmed that the predicted compounds significantly inhibited SARS-CoV-2 replication in Vero E6 cells at nanomolar, relatively non-toxic concentrations, with half-maximal inhibitory concentrations of 165.7 nM, 16.5 nM, and 31.4 nM for homoharringtonine, narciclasine, and anisomycin, respectively. Together, our results corroborate a host-centric strategy to inform protective antiviral therapies for COVID-19.

12.
EMBO Mol Med ; 13(1): e12828, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-914845

ABSTRACT

To circumvent the devastating pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a humanized decoy antibody (ACE2-Fc fusion protein) was designed to target the interaction between viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2). First, we demonstrated that ACE2-Fc could specifically abrogate virus replication by blocking the entry of SARS-CoV-2 spike-expressing pseudotyped virus into both ACE2-expressing lung cells and lung organoids. The impairment of viral entry was not affected by virus variants, since efficient inhibition was also observed in six SARS-CoV-2 clinical strains, including the D614G variants which have been shown to exhibit increased infectivity. The preservation of peptidase activity also enables ACE2-Fc to reduce the angiotensin II-mediated cytokine cascade. Furthermore, this Fc domain of ACE2-Fc was shown to activate NK cell degranulation after co-incubation with Spike-expressing H1975 cells. These promising characteristics potentiate the therapeutic prospects of ACE2-Fc as an effective treatment for COVID-19.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Viral/pharmacology , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Chlorocebus aethiops , HEK293 Cells , Host-Pathogen Interactions/drug effects , Humans , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Vero Cells
13.
Cell Rep ; 33(2): 108254, 2020 10 13.
Article in English | MEDLINE | ID: covidwho-812312

ABSTRACT

Development of specific antiviral agents is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infection. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validate cleavage at a putative furin substrate motif at SARS-CoV-2 spikes by expressing it in VeroE6 cells and find prominent syncytium formation. Cleavage and the syncytium are abolished by treatment with the furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein, but not by the transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein show antiviral effects on SARS-CoV-2-infected cells by decreasing virus production and cytopathic effects. Further analysis reveals that, similar to camostat, CMK blocks virus entry, but it further suppresses cleavage of spikes and the syncytium. Naphthofluorescein acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may be promising antiviral agents for prevention and treatment of SARS-CoV-2 infection.


Subject(s)
Amino Acid Chloromethyl Ketones/pharmacology , Antiviral Agents/pharmacology , Fluoresceins/pharmacology , Furin/antagonists & inhibitors , Protease Inhibitors/pharmacology , Spike Glycoprotein, Coronavirus/metabolism , Virus Replication , Animals , Betacoronavirus/drug effects , Betacoronavirus/metabolism , Betacoronavirus/physiology , Chlorocebus aethiops , Humans , Proteolysis , SARS-CoV-2 , Vero Cells
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