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1.
Bioconjug Chem ; 32(5): 1034-1046, 2021 05 19.
Article in English | MEDLINE | ID: covidwho-1217668

ABSTRACT

SARS-CoV-2 caused the COVID-19 pandemic that lasted for more than a year. Globally, there is an urgent need to use safe and effective vaccines for immunization to achieve comprehensive protection against SARS-CoV-2 infection. Focusing on developing a rapid vaccine platform with significant immunogenicity as well as broad and high protection efficiency, we designed a SARS-CoV-2 spike protein receptor-binding domain (RBD) displayed on self-assembled ferritin nanoparticles. In a 293i cells eukaryotic expression system, this candidate vaccine was prepared and purified. After rhesus monkeys are immunized with 20 µg of RBD-ferritin nanoparticles three times, the vaccine can elicit specific humoral immunity and T cell immune response, and the neutralizing antibodies can cross-neutralize four SARS-CoV-2 strains from different sources. In the challenge protection test, after nasal infection with 2 × 105 CCID50 SARS-CoV-2 virus, compared with unimmunized control animals, virus replication in the vaccine-immunized rhesus monkeys was significantly inhibited, and respiratory pathology observations also showed only slight pathological damage. These analyses will benefit the immunization program of the RBD-ferritin nanoparticle vaccine in the clinical trial design and the platform construction to present a specific antigen domain in the self-assembling nanoparticle in a short time to harvest stable, safe, and effective vaccine candidates for new SARS-CoV-2 isolates.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Nanoparticles/chemistry , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/immunology , Animals , Antibodies, Neutralizing/immunology , Binding Sites , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Ferritins/chemistry , Ferritins/metabolism , Immunity, Humoral , Macaca mulatta , Male , Nanoparticles/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/metabolism , Ultracentrifugation
2.
Cells ; 10(3)2021 03 04.
Article in English | MEDLINE | ID: covidwho-1125522

ABSTRACT

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators' safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.


Subject(s)
COVID-19/blood , Containment of Biohazards/methods , Extracellular Vesicles/chemistry , Virus Inactivation , COVID-19/virology , Detergents/pharmacology , Extracellular Vesicles/drug effects , Extracellular Vesicles/genetics , Hot Temperature , Humans , MicroRNAs/analysis , Microarray Analysis , Microscopy, Atomic Force , SARS-CoV-2 , Tetraspanins/analysis , Ultracentrifugation
3.
J Virol Methods ; 286: 113977, 2020 12.
Article in English | MEDLINE | ID: covidwho-800505

ABSTRACT

The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a ∼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the utility of PEG-mediated precipitation for SARS-CoV-2 and provides a resource for a range of coronavirus research areas.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , Polyethylene Glycols/chemistry , Animals , COVID-19 , COVID-19 Testing , Caco-2 Cells , Chlorocebus aethiops , Coronavirus Infections/diagnosis , Humans , Pandemics , Pneumonia, Viral/diagnosis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Ultracentrifugation/methods , Vero Cells
4.
Mem. Inst. Oswaldo Cruz ; 115: e200196, 2020. tab, graf
Article in English | WHO COVID, LILACS (Americas) | ID: covidwho-690981

ABSTRACT

This study presents preliminary results from a sewage-based surveillance to monitor the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the municipality of Niterói, State of Rio de Janeiro, Brazil. By using ultracentrifugation method associated to quantitative reverse transcription polymerase chain reaction (RT-qPCR) we detected SARS-CoV-2 in 41.6% (5/12) of raw sewage samples obtained from sewage treatment plants and sewers network in the city. This pioneer study carried out in Brazil aims to subsidise information for health surveillance concerning the viral circulation in different areas of the city and, revealed the insertion and importance of environmental virology in health public policies.


Subject(s)
Humans , Pneumonia, Viral/epidemiology , Sewage/virology , Coronavirus Infections/epidemiology , Betacoronavirus/isolation & purification , Ultracentrifugation , Brazil , Reverse Transcriptase Polymerase Chain Reaction , Pandemics , SARS-CoV-2 , COVID-19
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