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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-337621

ABSTRACT

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions is essential for development of new COVID-19 treatment strategies. Here we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. No changes to other cyclins or cyclin dependent kinases were observed. Further, cyclin D depletion was independent from SARS-CoV-2 mediated cell cycle arrest in early S phase or S/G2/M phase. Cyclin D3 knockdown by small interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 Envelope and Membrane proteins. We propose that cyclin D3 inhibits virion assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions.

2.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-335293

ABSTRACT

Background There are no real world data on vaccine elicited neutralising antibody responses for the world’s most widely used vaccine, AZD1222, in African populations following scale up. Here, we measured i) baseline SARS-CoV-2 seroprevalence and levels of protective neutralizing antibodies prior to vaccination rollout using both flow cytometric based analysis of binding antibodies to nucleocapsid (N), coupled with virus neutralisation approaches and ii) neutralizing antibody responses to VOC prior to vaccination (January 2021) and after two-doses of AZD1222 vaccine administered between June and July 2021 in Lagos, Nigeria - a period when the Delta variant was circulating. Methods Health workers at multiple sites in Lagos were recruited to the study. For binding antibody measurement, IgG antibodies against SARS-COV-2 Wuhan-1 receptor-binding domain (RBD), trimeric spike protein (S), nucleocapsid protein (N) and Omicron S1 were measured using the Luminex-based SARS-CoV-2-IgG assay by flow cytometry. For plasma neutralising antibody measurement, SARS-CoV-2 lentiviral pseudovirus (PV) were prepared by transfecting 293T cells with Wuhan-614G wild type (WT), B.1.617.2 (Delta) and BA.1 (Omicron) plasmids in conjunction with HIV-1 expression vectors and luciferase encoding genome flanked by LTRs. We performed serial plasma dilutions from each time point and mixed plasma with PV before infecting HeLa-ACE2 cell lines, reading out luminescence and calculating ID50 (reciprocal dilution of sera required to inhibit 50% of PV infection). Results Our study population receiving at least one dose of vaccine comprised 140 participants with a median age of 40 (interquartile range: 33, 48). 62/140 (44%) participants were anti-N IgG positive prior to administration of first vaccine dose. 49 had plasma samples available at baseline prior to vaccination and at two follow-up timepoints post vaccination for neutralization assays. Half of the participants, 25/49 (51%) were IgG anti-N positive at baseline. Of the 24 individuals anti-N Ab negative at baseline, 12/24 had ID 50 above the cut-off of 20. In these individuals, binding antibodies to S were also detectable, and neutralisation correlated with IgG anti-S, suggesting waning of N antibody after infection. Overall, neutralizing Ab titres to WT 1 month after second dose were 2579 and at 3 months post second-dose were 1695. As expected, lower levels of neutralization were observed against the Delta GMT 549 and Omicron variants 269 at 1 month. Positive anti-N IgG Ab status at baseline was associated with significantly higher titres of neutralizing antibodies following vaccination across all tested VOC. Those with anti-N Abs present at baseline did not experience waning of responses between months 1 and 3 post second dose. When data were analysed for negative anti-N IgG status at any timepoint, there was a significant decline in neutralization and binding antibodies between 1 month and 3 months post second-dose. The GMT in these individuals for Delta and Omicron was approximately 100, nearly a log lower in comparison to WT. We tested anti-N IgG in subjects who were anti-N IgG negative at baseline (n=78) and became positive between 1- and 3-months post second dose and found 7/49 (14%) with de-novo infection, with one additional participant demonstrating both reinfection and breakthrough infection to yield a total breakthrough rate of 8/49 (16%). Neutralising and binding Ab titres 1 month post vaccine, prior to breakthrough, were not associated with breakthrough infection. Neutralizing titres were higher at the last time point in individuals who had experienced vaccine breakthrough infection (with no evidence of infection prior to vaccine), indicating a boosting effect of infection in addition to vaccine. The increase in titres against Delta PV observed in breakthrough was significantly greater than the increase for WT and Omicron PVs, coincident with in the Delta wave of infection during the sampling period. Conclusions AZD1222 is immunogenic in this real orld west African cohort with significant background seroprevalence and incidence of breakthrough infection over a short time period. Prior infection and breakthrough infection induced higher anti-SARS-CoV-2 Ab responses at 3 months post vaccine against all widely circulating VOC. However, responses to Omicron BA.1 were low at three months regardless of prior exposure or breakthrough infection. Booster doses after AZD1222 should be considered for those at high risk in the African setting, even after natural infection, as future variants may be more pathogenic as well as immune evasive in the context of waning immunity.

3.
Nature ; 603(7902): 706-714, 2022 03.
Article in English | MEDLINE | ID: covidwho-1764186

ABSTRACT

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Subject(s)
COVID-19/pathology , COVID-19/virology , Membrane Fusion , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19 Vaccines/immunology , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Convalescence , Female , Humans , Immune Sera/immunology , Intestines/pathology , Intestines/virology , Lung/pathology , Lung/virology , Male , Middle Aged , Mutation , Nasal Mucosa/pathology , Nasal Mucosa/virology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Tissue Culture Techniques , Virulence , Virus Replication
4.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-317173

ABSTRACT

The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and has spread throughout India, displacing the B.1.1.7 (Alpha) variant and other pre-existing lineages. Mathematical modelling indicates that the growth advantage is most likely explained by a combination of increased transmissibility and immune evasion. Indeed in vitro, the delta variant is less sensitive to neutralising antibodies in sera from recovered individuals, with higher replication efficiency as compared to the Alpha variant. In an analysis of vaccine breakthrough in over 100 healthcare workers across three centres in India, the Delta variant not only dominates vaccine-breakthrough infections with higher respiratory viral loads compared to non-delta infections (Ct value of 16.5 versus 19), but also generates greater transmission between HCW as compared to B.1.1.7 or B.1.617.1 (p=0.02). In vitro, the Delta variant shows 8 fold approximately reduced sensitivity to vaccine-elicited antibodies compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against the SARS-CoV-2 Delta variant were significantly lower in participants vaccinated with ChadOx-1 as compared to BNT162b2 (GMT 3372 versus 654, p<0001). These combined epidemiological and in vitro data indicate that the dominance of the Delta variant in India has been most likely driven by a combination of evasion of neutralising antibodies in previously infected individuals and increased virus infectivity. Whilst severe disease in fully vaccinated HCW was rare, breakthrough transmission clusters in hospitals associated with the Delta variant are concerning and indicate that infection control measures need continue in the post-vaccination era.

5.
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.

6.
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
7.
Nature ; 592(7853): 277-282, 2021 04.
Article in English | MEDLINE | ID: covidwho-1387425

ABSTRACT

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.


Subject(s)
COVID-19/drug therapy , COVID-19/therapy , COVID-19/virology , Evolution, Molecular , Mutagenesis/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Aged , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Chronic Disease , Genome, Viral/drug effects , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing , Humans , Immune Evasion/drug effects , Immune Evasion/genetics , Immune Evasion/immunology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Immunization, Passive , Male , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutation , Phylogeny , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Time Factors , Viral Load/drug effects , Virus Shedding
8.
Cell Rep ; 35(13): 109292, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1281394

ABSTRACT

We report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike ΔH69/V70 in multiple independent lineages, often occurring after acquisition of receptor binding motif replacements such as N439K and Y453F, known to increase binding affinity to the ACE2 receptor and confer antibody escape. In vitro, we show that, although ΔH69/V70 itself is not an antibody evasion mechanism, it increases infectivity associated with enhanced incorporation of cleaved spike into virions. ΔH69/V70 is able to partially rescue infectivity of spike proteins that have acquired N439K and Y453F escape mutations by increased spike incorporation. In addition, replacement of the H69 and V70 residues in the Alpha variant B.1.1.7 spike (where ΔH69/V70 occurs naturally) impairs spike incorporation and entry efficiency of the B.1.1.7 spike pseudotyped virus. Alpha variant B.1.1.7 spike mediates faster kinetics of cell-cell fusion than wild-type Wuhan-1 D614G, dependent on ΔH69/V70. Therefore, as ΔH69/V70 compensates for immune escape mutations that impair infectivity, continued surveillance for deletions with functional effects is warranted.


Subject(s)
COVID-19/immunology , COVID-19/virology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cell Line , Chlorocebus aethiops , HEK293 Cells , Humans , Immune Evasion , Mutation , Pandemics , Phylogeny , Protein Binding , Recurrence , SARS-CoV-2/immunology , Vero Cells
9.
Cell Rep Med ; 1(6): 100099, 2020 09 22.
Article in English | MEDLINE | ID: covidwho-738567

ABSTRACT

Rapid COVID-19 diagnosis in the hospital is essential, although this is complicated by 30%-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant dominates the pandemic and it is unclear how serological tests designed to detect anti-spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild-type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95% CI 57.8-92.9) by rapid NAAT alone. The combined point of care antibody test and rapid NAAT is not affected by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Point-of-Care Testing , SARS-CoV-2/isolation & purification , Aged , Aged, 80 and over , Antibodies, Viral/blood , COVID-19 Testing/standards , Female , Humans , Immunoassay , Male , Middle Aged , Neutralization Tests , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
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