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1.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-295504

ABSTRACT

The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro , B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

2.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-294401

ABSTRACT

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients;b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays;a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM;a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins);a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG;a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

3.
Front Immunol ; 12: 748291, 2021.
Article in English | MEDLINE | ID: covidwho-1555236

ABSTRACT

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.


Subject(s)
COVID-19/immunology , Convalescence , Immunity, Humoral , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antigens, Viral/immunology , Biomarkers/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19 Serological Testing/standards , Calibration , Humans , Immunoglobulin Isotypes/blood , Immunoglobulin Isotypes/immunology , Reference Standards , Severity of Illness Index
4.
Nature ; 599(7883): 114-119, 2021 11.
Article in English | MEDLINE | ID: covidwho-1392870

ABSTRACT

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.


Subject(s)
Immune Evasion , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Virus Replication/immunology , Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , Cell Fusion , Cell Line , Female , Health Personnel , Humans , India , Kinetics , Male , Spike Glycoprotein, Coronavirus/metabolism , Vaccination
5.
FEBS Lett ; 595(18): 2323-2340, 2021 09.
Article in English | MEDLINE | ID: covidwho-1332924

ABSTRACT

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Receptors, Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Bacterial Proteins/chemistry , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/chemistry , DNA-Binding Proteins/chemistry , Ferritins/chemistry , Humans , Immunization , Mice , Nanoparticles , Protein Domains , Protein Multimerization , Spike Glycoprotein, Coronavirus/chemistry , Sulfolobus
6.
EMBO J ; 40(17): e108588, 2021 09 01.
Article in English | MEDLINE | ID: covidwho-1332432

ABSTRACT

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T-cell activity correlates within individuals, suggesting N antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.


Subject(s)
Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/isolation & purification , COVID-19/blood , SARS-CoV-2/isolation & purification , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/virology , Humans , Nucleoproteins/blood , Nucleoproteins/immunology , SARS-CoV-2/pathogenicity
7.
PLoS Pathog ; 17(1): e1009246, 2021 01.
Article in English | MEDLINE | ID: covidwho-1045566

ABSTRACT

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor ACE2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors may reduce but not abolish viral spread.


Subject(s)
Cell Fusion , Furin/genetics , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Animals , COVID-19 , CRISPR-Cas Systems , Chlorocebus aethiops , Gene Knockout Techniques , HEK293 Cells , Humans , Protein Structure, Tertiary , SARS-CoV-2 , Serine Endopeptidases , Vero Cells
8.
Cell Stem Cell ; 27(6): 951-961.e5, 2020 12 03.
Article in English | MEDLINE | ID: covidwho-857180

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain organoids to examine SARS-CoV-2 neurotropism. We find expression of viral receptor ACE2 in mature choroid plexus cells expressing abundant lipoproteins, but not in neurons or other cell types. We challenge organoids with SARS-CoV-2 spike pseudovirus and live virus to demonstrate viral tropism for choroid plexus epithelial cells but little to no infection of neurons or glia. We find that infected cells are apolipoprotein- and ACE2-expressing cells of the choroid plexus epithelial barrier. Finally, we show that infection with SARS-CoV-2 damages the choroid plexus epithelium, leading to leakage across this important barrier that normally prevents entry of pathogens, immune cells, and cytokines into cerebrospinal fluid and the brain.


Subject(s)
Blood-Brain Barrier/virology , Choroid Plexus/virology , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Models, Biological , Organoids/virology , Vero Cells , Viral Tropism , Virus Internalization
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