Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 167
Filter
1.
BMJ Open ; 12(4): e056872, 2022 Apr 08.
Article in English | MEDLINE | ID: covidwho-1784824

ABSTRACT

OBJECTIVE: Assessing safety and immunogenicity of an inactivated whole virus particle vaccine. DESIGN: Single-centre, double-blind, randomised, placebo-controlled, phase I (stage I: 18-50, stage II: 51-75 years), phase II (18-75 years) clinical trials. SETTING: 29 December 2020 to 22 April 2021. PARTICIPANTS: Stage I-phase I: 56 participants; stage II-phase I: 32; phase II: 280. INTERVENTION: During stage I, participants randomly (3:3:1) received 3 µg, 5 µg vaccine or placebo in a 14-day interval. Participants in stage II received two shots of 5 µg vaccine or placebo (3:1). In phase II, participants received 5 µg vaccine or placebo (4:1) in a 28-day interval. PRIMARY AND SECONDARY OUTCOME MEASURES: Safety assessment and immunogenicity assessment via antibody response and conventional virus neutralisation test (cVNT). RESULTS: All adverse events (AEs) were mild or moderate and transient in both phase I and phase II, and no AEs of special interest were reported. The seroconversion-rate of neutralising, antireceptor binding-domain (RBD) and anti-spike-glycoprotein (anti-S) antibodies 14-days after second dose of 5 µg vaccine in stage I was 70.8% (95% CI 48.9% to 87.4%), 87.5% (95% CI 67.6% to 97.3%), 91.7% (95% CI 73.0% to 99.0%). The antibody titres increased more among 5 µg than 3 µg. The corresponding rates for 3 µg vaccine were 45.8% (95% CI 25.6% to 67.2%), 54.2% (95% CI 32.8% to 74.5%) and 70.8% (95% CI 48.9% to 87.4%), respectively. In stage II, 100% (95% CI 84.6% to 100%), 86.4% (95% CI 65.1% to 97.1%) and 86.4% (95% CI 65.1% to 97.1%) of participants seroconverted for neutralising, anti-RBD and anti-S antibodies. In phase II, the seroconversion rate of neutralising-antibody was 82.8% (95% CI 77.0% to 87.6%), anti-RBD 77.0% (95% CI 70.7% to 82.6%) and anti-S 79.9% (95% CI 73.8% to 85.1%) on day 42. In the cVNT, the sera at 1/64 times dilution would neutralise SARS-CoV-2 among 91.7%, 77.3% and 82.5% of vaccinated participants in phase I-stage I, phase I-stage II and phase II clinical trials, respectively. CONCLUSIONS: These results support further evaluation of this inactivated whole virus particle vaccine. TRIAL REGISTRATION NUMBERS: IRCT20201202049567N1 and IRCT20201202049567N2 for phase I and IRCT20201202049567N3 for phase II.


Subject(s)
COVID-19 , SARS-CoV-2 , Adult , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Double-Blind Method , Humans , Vaccines, Inactivated/adverse effects , Virion
2.
Viruses ; 14(3)2022 03 18.
Article in English | MEDLINE | ID: covidwho-1765956

ABSTRACT

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100-200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production.


Subject(s)
HIV-1 , Viruses, Unclassified , Animals , Baculoviridae/genetics , DNA , HEK293 Cells , HIV-1/genetics , Humans , Mammals , Virion/genetics , Viruses, Unclassified/genetics
3.
J Virol ; 96(2): e0106021, 2022 01 26.
Article in English | MEDLINE | ID: covidwho-1759286

ABSTRACT

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.


Subject(s)
Capsid/chemistry , Mutation/drug effects , Rhinovirus/physiology , Virus Uncoating/physiology , Antiviral Agents/pharmacology , Capsid/drug effects , Capsid Proteins/genetics , Capsid Proteins/metabolism , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Endosomes/chemistry , Endosomes/drug effects , Endosomes/metabolism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Intercellular Adhesion Molecule-1/metabolism , Protein Conformation , Rhinovirus/chemistry , Rhinovirus/drug effects , Rhinovirus/genetics , Virion/chemistry , Virion/genetics , Virion/metabolism , Virus Internalization/drug effects , Virus Uncoating/drug effects , Virus Uncoating/genetics
4.
PLoS Pathog ; 18(2): e1010268, 2022 02.
Article in English | MEDLINE | ID: covidwho-1753212

ABSTRACT

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.


Subject(s)
Ebolavirus/genetics , Filoviridae Infections/virology , Filoviridae/genetics , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Genetic Complementation Test , Genome, Viral , Hemorrhagic Fever, Ebola/virology , Host Microbial Interactions , Humans , Inclusion Bodies/virology , Induced Pluripotent Stem Cells/virology , Macrophages/virology , RNA, Viral , Reverse Genetics , Vero Cells , Virion/genetics
5.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1715774

ABSTRACT

Virus-like particles resemble infectious virus particles in size, shape, and molecular composition; however, they fail to productively infect host cells. Historically, the presence of virus-like particles has been inferred from total particle counts by microscopy, and infectious particle counts or plaque-forming-units (PFUs) by plaque assay; the resulting ratio of particles-to-PFUs is often greater than one, easily 10 or 100, indicating that most particles are non-infectious. Despite their inability to hijack cells for their reproduction, virus-like particles and the defective genomes they carry can exhibit a broad range of behaviors: interference with normal virus growth during co-infections, cell killing, and activation or inhibition of innate immune signaling. In addition, some virus-like particles become productive as their multiplicities of infection increase, a sign of cooperation between particles. Here, we review established and emerging methods to count virus-like particles and characterize their biological functions. We take a critical look at evidence for defective interfering virus genomes in natural and clinical isolates, and we review their potential as antiviral therapeutics. In short, we highlight an urgent need to better understand how virus-like genomes and particles interact with intact functional viruses during co-infection of their hosts, and their impacts on the transmission, severity, and persistence of virus-associated diseases.


Subject(s)
Defective Viruses/physiology , Virion/physiology , Animals , Colony-Forming Units Assay , Genome, Viral , Humans , Microscopy, Electron, Transmission , Viral Plaque Assay , Virus Diseases/virology , Virus Replication
6.
Viruses ; 14(2)2022 01 25.
Article in English | MEDLINE | ID: covidwho-1715763

ABSTRACT

Epithelial cells are apico-basolateral polarized cells that line all tubular organs and are often targets for infectious agents. This review focuses on the release of human RNA virus particles from both sides of polarized human cells grown on transwells. Most viruses that infect the mucosa leave their host cells mainly via the apical side while basolateral release is linked to virus propagation within the host. Viruses do this by hijacking the cellular factors involved in polarization and trafficking. Thus, understanding epithelial polarization is essential for a clear understanding of virus pathophysiology.


Subject(s)
Epithelial Cells/virology , RNA Viruses/physiology , Virus Release , Cell Polarity , Humans , Virion/physiology , Virus Assembly , Virus Replication
7.
Med Mol Morphol ; 55(1): 60-67, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1712248

ABSTRACT

SARS-CoV-2 is the cause of COVID-19. The three-dimensional morphology of viral particles existing and multiplying in infected cells has not been established by electron tomography, which is different from cryo-electron tomography using frozen samples. In this study, we establish the morphological structure of SARS-CoV-2 particles by three-dimensional reconstruction of images obtained by electron tomography and transmission electron microscopy of biological samples embedded in epoxy resin. The characteristic roots of spike structures were found to be arranged at the surface of a virion covered with an envelope. A high-electron-density structure that appears to be a nucleocapsid was observed inside the envelope of the virion on three-dimensional images reconstructed by electron tomography. The SARS-CoV-2 particles that budded in the vacuoles in the cytoplasm were morphologically identical to those found outside the cells, suggesting that mature and infectious SARS-CoV-2 particles were already produced in the vacuoles. Here, we show the three-dimensional morphological structure of SARS-CoV-2 particles reconstructed by electron tomography. To control infection, inhibition of viral release from vacuoles would be a new target in the development of prophylactic agents against SARS-CoV-2.


Subject(s)
Electron Microscope Tomography , SARS-CoV-2 , COVID-19 , Humans , Imaging, Three-Dimensional , SARS-CoV-2/ultrastructure , Virion/ultrastructure
8.
Sci Rep ; 12(1): 1724, 2022 02 02.
Article in English | MEDLINE | ID: covidwho-1699378

ABSTRACT

This study introduces localized surface plasmon resonance (L-SPR) mediated heating filter membrane (HFM) for inactivating universal viral particles by using the photothermal effect of plasmonic metal nanoparticles (NPs). Plasmonic metal NPs were coated onto filter membrane via a conventional spray-coating method. The surface temperature of the HFM could be controlled to approximately 40-60 °C at room temperature, owing to the photothermal effect of the gold (Au) NPs coated on them, under irradiation by visible light-emitting diodes. Due to the photothermal effect of the HFMs, the virus titer of H1Npdm09 was reduced by > 99.9%, the full inactivation time being < 10 min, confirming the 50% tissue culture infective dose (TCID50) assay. Crystal violet staining showed that the infectious samples with photothermal inactivation lost their infectivity against Mardin-Darby Canine Kidney cells. Moreover, photothermal inactivation could also be applied to reduce the infectivity of SARS-CoV-2, showing reduction rate of 99%. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to confirm the existence of viral genes on the surface of the HFM. The results of the TCID50 assay, crystal violet staining method, and qRT-PCR showed that the effective and immediate reduction in viral infectivity possibly originated from the denaturation or deformation of membrane proteins and components. This study provides a new, simple, and effective method to inactivate viral infectivity, leading to its potential application in various fields of indoor air quality control and medical science.


Subject(s)
COVID-19/virology , Hot Temperature , Light , Metal Nanoparticles , Micropore Filters , SARS-CoV-2 , Surface Plasmon Resonance/methods , Virion , Virus Inactivation , Air Pollution, Indoor , Animals , Cells, Cultured , Dogs , Gold/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity
9.
Nat Commun ; 13(1): 868, 2022 02 14.
Article in English | MEDLINE | ID: covidwho-1684025

ABSTRACT

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.


Subject(s)
COVID-19/immunology , Fatty Acids/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology , A549 Cells , Allosteric Site/genetics , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Fatty Acid-Binding Proteins/immunology , Fatty Acid-Binding Proteins/metabolism , Fatty Acids/metabolism , Humans , MCF-7 Cells , Microscopy, Confocal/methods , Protein Binding , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virion/ultrastructure
10.
Life Sci ; 295: 120411, 2022 Apr 15.
Article in English | MEDLINE | ID: covidwho-1683412

ABSTRACT

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.


Subject(s)
COVID-19/drug therapy , Deoxyglucose/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Adenosine Triphosphate/metabolism , Animals , Antiviral Agents/pharmacology , COVID-19/metabolism , COVID-19/virology , Cell Proliferation/drug effects , Cell Survival/drug effects , Chlorocebus aethiops , Glucose/metabolism , Glycolysis/drug effects , Glycosylation , Host-Pathogen Interactions/drug effects , Mannose/pharmacology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virion/drug effects , Virion/pathogenicity , Virus Replication/drug effects
11.
Cells ; 11(3)2022 01 20.
Article in English | MEDLINE | ID: covidwho-1643582

ABSTRACT

Pathogenic enveloped viruses are covered with a glycan shield that provides a dual function: the glycan structures contribute to virus protection as well as host cell recognition. The three classical types of N-glycans, in particular complex glycans, high-mannose glycans, and hybrid glycans, together with some O-glycans, participate in the glycan shield of the Ebola virus, influenza virus, human cytomegalovirus, herpes virus, human immunodeficiency virus, Lassa virus, and MERS-CoV, SARS-CoV, and SARS-CoV-2, which are responsible for respiratory syndromes. The glycans are linked to glycoproteins that occur as metastable prefusion glycoproteins on the surface of infectious virions such as gp120 of HIV, hemagglutinin of influenza, or spike proteins of beta-coronaviruses. Plant lectins with different carbohydrate-binding specificities and, especially, mannose-specific lectins from the Vicieae tribe, such as pea lectin and lentil lectin, can be used as glycan probes for targeting the glycan shield because of their specific interaction with the α1,6-fucosylated core Man3GlcNAc2, which predominantly occurs in complex and hybrid glycans. Other plant lectins with Neu5Ac specificity or GalNAc/T/Tn specificity can also serve as potential glycan probes for the often sialylated complex glycans and truncated O-glycans, respectively, which are abundantly distributed in the glycan shield of enveloped viruses. The biomedical and therapeutical potential of plant lectins as antiviral drugs is discussed.


Subject(s)
COVID-19/metabolism , Fabaceae/metabolism , Plant Lectins/metabolism , Polysaccharides/metabolism , SARS-CoV-2/metabolism , Viral Envelope/metabolism , COVID-19/epidemiology , COVID-19/virology , Humans , Mannose/metabolism , Protein Binding , SARS-CoV-2/physiology , Virion/metabolism , Virus Internalization
12.
Biochim Biophys Acta Mol Basis Dis ; 1868(4): 166347, 2022 04 01.
Article in English | MEDLINE | ID: covidwho-1636951

ABSTRACT

As epitomised by the COVID-19 pandemic, diseases caused by viruses are one of the greatest health and economic burdens to human society. Viruses are 'nanostructures', and their small size (typically less than 200 nm in diameter) can make it challenging to obtain images of their morphology and structure. Recent advances in fluorescence microscopy have given rise to super-resolution techniques, which have enabled the structure of viruses to be visualised directly at a resolution in the order of 20 nm. This mini-review discusses how recent state-of-the-art super-resolution imaging technologies are providing new nanoscale insights into virus structure.


Subject(s)
Microscopy, Fluorescence , Viruses/chemistry , Humans , Imaging, Three-Dimensional , Virion/chemistry
13.
J Virol ; 96(6): e0189721, 2022 03 23.
Article in English | MEDLINE | ID: covidwho-1631836

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2' sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike's conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2' cleavage sites in S protein synthesis and processing as well as virus assembly and infection. IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2' sites are very important, like the S2' cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virus Internalization
14.
Viruses ; 14(1)2022 01 08.
Article in English | MEDLINE | ID: covidwho-1614009

ABSTRACT

Photodynamic inactivation (PDI) employs a photosensitizer, light, and oxygen to create a local burst of reactive oxygen species (ROS) that can inactivate microorganisms. The botanical extract PhytoQuinTM is a powerful photosensitizer with antimicrobial properties. We previously demonstrated that photoactivated PhytoQuin also has antiviral properties against herpes simplex viruses and adenoviruses in a dose-dependent manner across a broad range of sub-cytotoxic concentrations. Here, we report that human coronaviruses (HCoVs) are also susceptible to photodynamic inactivation. Photoactivated-PhytoQuin inhibited the replication of the alphacoronavirus HCoV-229E and the betacoronavirus HCoV-OC43 in cultured cells across a range of sub-cytotoxic doses. This antiviral effect was light-dependent, as we observed minimal antiviral effect of PhytoQuin in the absence of photoactivation. Using RNase protection assays, we observed that PDI disrupted HCoV particle integrity allowing for the digestion of viral RNA by exogenous ribonucleases. Using lentiviruses pseudotyped with the SARS-CoV-2 Spike (S) protein, we once again observed a strong, light-dependent antiviral effect of PhytoQuin, which prevented S-mediated entry into human cells. We also observed that PhytoQuin PDI altered S protein electrophoretic mobility. The PhytoQuin constituent emodin displayed equivalent light-dependent antiviral activity to PhytoQuin in matched-dose experiments, indicating that it plays a central role in PhytoQuin PDI against CoVs. Together, these findings demonstrate that HCoV lipid envelopes and proteins are damaged by PhytoQuin PDI and expands the list of susceptible viruses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Photosensitizing Agents/pharmacology , Virus Inactivation/drug effects , Animals , Antiviral Agents/radiation effects , Cell Line , Cell Survival/drug effects , Cricetinae , Emodin/pharmacology , Emodin/radiation effects , Humans , Light , Photosensitizing Agents/radiation effects , Plant Extracts/pharmacology , Plant Extracts/radiation effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/drug effects , Virion/drug effects
15.
JAMA Netw Open ; 5(1): e2142210, 2022 01 04.
Article in English | MEDLINE | ID: covidwho-1611175

ABSTRACT

Importance: A surge of COVID-19 occurred from March to June 2021, in New Delhi, India, linked to the B.1.617.2 (Delta) variant of SARS-CoV-2. COVID-19 vaccines were rolled out for health care workers (HCWs) starting in January 2021. Objective: To assess the incidence density of reinfection among a cohort of HCWs and estimate the effectiveness of the inactivated whole virion vaccine BBV152 against reinfection. Design, Setting, and Participants: This was a retrospective cohort study among HCWs working at a tertiary care center in New Delhi, India. Exposures: Vaccination with 0, 1, or 2 doses of BBV152. Main Outcomes and Measures: The HCWs were categorized as fully vaccinated (with 2 doses and ≥15 days after the second dose), partially vaccinated (with 1 dose or 2 doses with <15 days after the second dose), or unvaccinated. The incidence density of COVID-19 reinfection per 100 person-years was computed, and events from March 3, 2020, to June 18, 2021, were included for analysis. Unadjusted and adjusted hazard ratios (HRs) were estimated using a Cox proportional hazards model. Estimated vaccine effectiveness (1 - adjusted HR) was reported. Results: Among 15 244 HCWs who participated in the study, 4978 (32.7%) were diagnosed with COVID-19. The mean (SD) age was 36.6 (10.3) years, and 55.0% were male. The reinfection incidence density was 7.26 (95% CI: 6.09-8.66) per 100 person-years (124 HCWs [2.5%], total person follow-up period of 1696 person-years as time at risk). Fully vaccinated HCWs had lower risk of reinfection (HR, 0.14 [95% CI, 0.08-0.23]), symptomatic reinfection (HR, 0.13 [95% CI, 0.07-0.24]), and asymptomatic reinfection (HR, 0.16 [95% CI, 0.05-0.53]) compared with unvaccinated HCWs. Accordingly, among the 3 vaccine categories, reinfection was observed in 60 of 472 (12.7%) of unvaccinated (incidence density, 18.05 per 100 person-years; 95% CI, 14.02-23.25), 39 of 356 (11.0%) of partially vaccinated (incidence density 15.62 per 100 person-years; 95% CI, 11.42-21.38), and 17 of 1089 (1.6%) fully vaccinated (incidence density 2.18 per 100 person-years; 95% CI, 1.35-3.51) HCWs. The estimated effectiveness of BBV152 against reinfection was 86% (95% CI, 77%-92%); symptomatic reinfection, 87% (95% CI, 76%-93%); and asymptomatic reinfection, 84% (95% CI, 47%-95%) among fully vaccinated HCWs. Partial vaccination was not associated with reduced risk of reinfection. Conclusions and Relevance: These findings suggest that BBV152 was associated with protection against both symptomatic and asymptomatic reinfection in HCWs after a complete vaccination schedule, when the predominant circulating variant was B.1.617.2.


Subject(s)
COVID-19/epidemiology , Health Personnel , Reinfection , SARS-CoV-2 , Adult , COVID-19/etiology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , Cohort Studies , Female , Humans , Immunogenicity, Vaccine , India/epidemiology , Male , Middle Aged , Surveys and Questionnaires , Tertiary Care Centers , Vaccines, Inactivated/administration & dosage , Virion/immunology , Young Adult
16.
Lancet ; 398(10296): 213-222, 2021 07 17.
Article in English | MEDLINE | ID: covidwho-1598580

ABSTRACT

BACKGROUND: CoronaVac, an inactivated whole-virion SARS-CoV-2 vaccine, has been shown to be well tolerated with a good safety profile in individuals aged 18 years and older in phase 1/2 trials, and provided a good humoral response against SARS-CoV-2. We present the interim efficacy and safety results of a phase 3 clinical trial of CoronaVac in Turkey. METHODS: This was a double-blind, randomised, placebo-controlled phase 3 trial. Volunteers aged 18-59 years with no history of COVID-19 and with negative PCR and antibody test results for SARS-CoV-2 were enrolled at 24 centres in Turkey. Exclusion criteria included (but were not limited to) immunosuppressive therapy (including steroids) within the past 6 months, bleeding disorders, asplenia, and receipt of any blood products or immunoglobulins within the past 3 months. The K1 cohort consisted of health-care workers (randomised in a 1:1 ratio), and individuals other than health-care workers were also recruited into the K2 cohort (randomised in a 2:1 ratio) using an interactive web response system. The study vaccine was 3 µg inactivated SARS-CoV-2 virion adsorbed to aluminium hydroxide in a 0·5 mL aqueous suspension. Participants received either vaccine or placebo (consisting of all vaccine components except inactivated virus) intramuscularly on days 0 and 14. The primary efficacy outcome was the prevention of PCR-confirmed symptomatic COVID-19 at least 14 days after the second dose in the per protocol population. Safety analyses were done in the intention-to-treat population. This study is registered with ClinicalTrials.gov (NCT04582344) and is active but no longer recruiting. FINDINGS: Among 11 303 volunteers screened between Sept 14, 2020, and Jan 5, 2021, 10 218 were randomly allocated. After exclusion of four participants from the vaccine group because of protocol deviations, the intention-to-treat group consisted of 10 214 participants (6646 [65·1%] in the vaccine group and 3568 [34·9%] in the placebo group) and the per protocol group consisted of 10 029 participants (6559 [65·4%] and 3470 [34·6%]) who received two doses of vaccine or placebo. During a median follow-up period of 43 days (IQR 36-48), nine cases of PCR-confirmed symptomatic COVID-19 were reported in the vaccine group (31·7 cases [14·6-59·3] per 1000 person-years) and 32 cases were reported in the placebo group (192·3 cases [135·7-261·1] per 1000 person-years) 14 days or more after the second dose, yielding a vaccine efficacy of 83·5% (95% CI 65·4-92·1; p<0·0001). The frequencies of any adverse events were 1259 (18·9%) in the vaccine group and 603 (16·9%) in the placebo group (p=0·0108) with no fatalities or grade 4 adverse events. The most common systemic adverse event was fatigue (546 [8·2%] participants in the vaccine group and 248 [7·0%] the placebo group, p=0·0228). Injection-site pain was the most frequent local adverse event (157 [2·4%] in the vaccine group and 40 [1·1%] in the placebo group, p<0·0001). INTERPRETATION: CoronaVac has high efficacy against PCR-confirmed symptomatic COVID-19 with a good safety and tolerability profile. FUNDING: Turkish Health Institutes Association.


Subject(s)
Antibodies, Neutralizing , COVID-19 Vaccines/therapeutic use , COVID-19/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/prevention & control , Double-Blind Method , Health Personnel/statistics & numerical data , Humans , Male , Middle Aged , Turkey , Vaccination , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Virion/immunology
18.
Analyst ; 147(2): 213-222, 2022 Jan 17.
Article in English | MEDLINE | ID: covidwho-1585755

ABSTRACT

The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 µL sample with a pipette. The device, which we call the µSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2-3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus (n = 62). Moreover, the dynamic range of the sensor extends at least from 105.9 virions per mL to 1010.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (105.6-107 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.


Subject(s)
COVID-19 , Pandemics , Humans , SARS-CoV-2 , Sensitivity and Specificity , Silicon , Virion
19.
J Chem Inf Model ; 62(1): 176-186, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1575860

ABSTRACT

The coronavirus disease 19 (COVID-19) pandemic is causing a global health crisis and has already caused a devastating societal and economic burden. The pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has a high sequence and architecture identity with SARS-CoV, but far more people have been infected by SARS-CoV-2. Here, combining the structural data from cryo-electron microscopy and structure prediction, we constructed bottom-up Martini coarse-grained models of intact SARS-CoV and SARS-CoV-2 envelopes. Microsecond molecular dynamics simulations were performed, allowing us to explore their dynamics and supramolecular organization. Both SARS-CoV and SARS-CoV-2 envelopes present a spherical morphology, with structural proteins forming multiple string-like islands in the membrane and clusters between the heads of spike proteins. Critical differences between the SARS-CoV and SARS-CoV-2 envelopes are the interaction pattern between the spike proteins and the flexibility of the spike proteins. Our models provide structural and dynamic insights into the SARS virus envelopes and could be used for further investigation, such as drug design and membrane fusion and fission processes.


Subject(s)
COVID-19 , SARS Virus , Cryoelectron Microscopy , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Viral Envelope , Virion
20.
J Cell Mol Med ; 26(1): 25-34, 2022 01.
Article in English | MEDLINE | ID: covidwho-1570773

ABSTRACT

Transmission electron microscopy has historically been indispensable for virology research, as it offers unique insight into virus function. In the past decade, as cryo-electron microscopy (cryo-EM) has matured and become more accessible, we have been able to peer into the structure of viruses at the atomic level and understand how they interact with the host cell, with drugs or with antibodies. Perhaps, there was no time in recent history where cryo-EM was more needed, as SARS-CoV-2 has spread around the globe, causing millions of deaths and almost unquantifiable economic devastation. In this concise review, we aim to mark the most important contributions of cryo-EM to understanding the structure and function of SARS-CoV-2 proteins, from surface spikes to the virus core and from virus-receptor interactions to antibody binding.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Antibodies, Viral/chemistry , COVID-19 Vaccines/chemistry , COVID-19/prevention & control , Receptors, Virus/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/biosynthesis , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/biosynthesis , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Humans , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Virus/immunology , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , SARS-CoV-2/ultrastructure , Serine Endopeptidases/chemistry , Serine Endopeptidases/immunology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Virion/drug effects , Virion/pathogenicity , Virion/ultrastructure
SELECTION OF CITATIONS
SEARCH DETAIL