SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion methods
Aim. Evidence-backed execution summary for SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion methods from SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.
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What do I need before I start?
human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Delta variant and neutralizing antibodies
reagent used in the protocol.
- Use
- We first plotted the relative proportion of variants in new cases of SARS-CoV-2 in India since the start of 2021. Although B.1.617.1 emerged earlier, the Delta variant B.1.617.2 has become more dominant (Fig. ). We hypothesized that B.1.617.2 would exhibit immune evasion to antibody responses generated by previous S...
Delta variant and neutralizing antibodies
reagent used in the protocol.
- Use
- We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChAdOx1 or BNT162b2. These experiments showed a loss of sensitivity for B.1.617.2 compared with WT Wuhan-1 bearing D614G of around eightfold fo...
Delta variant and neutralizing antibodies
reagent used in the protocol.
- Use
- We investigated the role of the B.1.617.2 spike as an escape mechanism by testing 33 spike-specific monoclonal antibodies with an in vitro PV neutralization assay using Vero E6 target cells expressing transmembrane protease serine 2 (TMPRSS2) and the Wuhan-1 D614G SARS-CoV-2 spike or the B.1.617.2 spike (Extend...
SARS-CoV-2 Delta variant replication
reagent used in the protocol.
- Use
- We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase in released virions from cells (Fig. ). Next we tested B.1.1.7 against two separate isolates of B.1.617.2 in a human airway epithelial (HAE...
B.1.617.2 spike-mediated cell entry
reagent used in the protocol.
- Use
- We tested single-round viral entry of B.1.617.1 and B.1.617.2 spike (Fig. and Extended Data Fig. ) using the PV system, infecting Calu-3 lung cells expressing endogenous levels of angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 (Fig. ), as well as other cells transduced or transiently transfected with ACE2 and TM...
PV experiments
reagent used in the protocol.
- Use
- HEK 293T CRL-3216, HeLa-ACE2 (gift from James Voss) and Vero CCL-81 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U ml -1 penicillin and 100 mg ml -1 streptomycin. All cells were regularly tested and found...
PV preparation for testing against vaccine-elicited antibodies and cell entry
reagent used in the protocol.
- Use
- Plasmids encoding the spike protein of SARS-CoV-2 D614 with a carboxy-terminal 19-amino-acid deletion with D614G were used. Mutations were introduced using the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent) following the manufacturer's instructions. Preparation of the B.1.1.7 S-expressing plasmid...
Standardization of virus input by SYBR Green-based product-enhanced PCR assay
reagent used in the protocol.
- Use
- The reverse transcriptase (RT) activity of virus preparations was determined by quantitative PCR (qPCR) using a SYBR Green-based product-enhanced PCR assay as previously described. In brief, tenfold dilutions of virus supernatant were lysed in a 1:1 ratio in a 2 × lysis solution (made up of 40% glycerol (v/v),...
Methods
Ethical approval for the study of vaccine-elicited antibodies in sera from vaccinees was obtained from the East of England - Cambridge Central Research Ethics Committee Cambridge (REC ref. 17/EE/0025). Use of convalescent sera had ethical approval from the South Central - Berkshire B Research Ethics Committee...
- Use
- Ethical approval for the study of vaccine-elicited antibodies in sera from vaccinees was obtained from the East of England - Cambridge Central Research Ethics Committee Cambridge (REC ref. 17/EE/0025). Use of convalescent sera had ethical approval from the South Central - Berkshire B Research Ethics Committee...
Delta variant and neutralizing antibodies
We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChAdOx1 or BNT162b2. These experiments showed a loss of sensitivity for B.1.617.2 compared with WT Wuhan-1 bearing D614G of around eightfold fo...
- Use
- We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChAdOx1 or BNT162b2. These experiments showed a loss of sensitivity for B.1.617.2 compared with WT Wuhan-1 bearing D614G of around eightfold fo...
SARS-CoV-2 Delta variant replication
We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase in released virions from cells (Fig. ). Next we tested B.1.1.7 against two separate isolates of B.1.617.2 in a human airway epithelial (HAE...
- Use
- We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase in released virions from cells (Fig. ). Next we tested B.1.1.7 against two separate isolates of B.1.617.2 in a human airway epithelial (HAE...
B.1.617.2 spike-mediated cell fusion
The plasma membrane route of entry, and indeed transmissibility in animal models, is critically dependent on the polybasic cleavage site between S1 and S2 (refs.,, ) and cleavage of spike before virion release from producer cells. Alterations at P681 in the polybasic cleavage site have been observed in mult...
- Use
- The plasma membrane route of entry, and indeed transmissibility in animal models, is critically dependent on the polybasic cleavage site between S1 and S2 (refs.,, ) and cleavage of spike before virion release from producer cells. Alterations at P681 in the polybasic cleavage site have been observed in mult...
B.1.617.2 spike-mediated cell entry
We tested single-round viral entry of B.1.617.1 and B.1.617.2 spike (Fig. and Extended Data Fig. ) using the PV system, infecting Calu-3 lung cells expressing endogenous levels of angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 (Fig. ), as well as other cells transduced or transiently transfected with ACE2 and TM...
- Use
- We tested single-round viral entry of B.1.617.1 and B.1.617.2 spike (Fig. and Extended Data Fig. ) using the PV system, infecting Calu-3 lung cells expressing endogenous levels of angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 (Fig. ), as well as other cells transduced or transiently transfected with ACE2 and TM...
B.1.617.2 vaccine breakthrough infection
We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs) started in early 2021 with the ChAdOx1 vaccine (Covishield). During the wave of infections in March and April, symptomatic SARS-CoV-2 was co...
- Use
- We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs) started in early 2021 with the ChAdOx1 vaccine (Covishield). During the wave of infections in March and April, symptomatic SARS-CoV-2 was co...
Sequencing quality control and phylogenetic analysis
Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then aligned to the reference strain MN908947.3 (Wuhan-Hu-1) with mafft v4.487 (ref. ) using the --keeplength and --addfragments options....
- Use
- Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then aligned to the reference strain MN908947.3 (Wuhan-Hu-1) with mafft v4.487 (ref. ) using the --keeplength and --addfragments options....
Sequencing quality control and phylogenetic analysis
Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were annotated in R v4.1.0 using ggtree v3.0.2 (ref. ) and rooted on the SARS-CoV-2 reference sequence ( MN908947.3 ). Nodes were arranged...
- Use
- Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were annotated in R v4.1.0 using ggtree v3.0.2 (ref. ) and rooted on the SARS-CoV-2 reference sequence ( MN908947.3 ). Nodes were arranged...
Vaccinee serum neutralization, live virus assays
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Vero E6 ACE2/TMPRSS2 cells were seeded at a cell density of 2 × 10 4 per well in a 96-well plate 24 h before infection. Serum was titrated starting at a final 1:10 dilution, with WT (SARS-CoV-2/human/Liverpool/REMRQ0001/2020), B.1.1.7 or B.1.617.2 virus isolates being added at an MOI of 0...
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Open the source paper before finalizing run-specific details.
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Open quote workflowStep-by-step procedure
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Extended data figures and tables
Extended Data Table 4 Relative recei1 vaccine effectiveness against B1.617.2 v non- B1.617.2: Upper Table: Odds ratios for detection of B.1.617.2 relative to non-B.1.617.2 in vaccinated compared to unvaccinated individuals in multi-variable logistic regression
SARS-CoV-2 Delta variant replication
We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase in released virions from cells (Fig. ). Next we tested B.1.1.7 against two separate isolates of B.1.617.2 in a human airway epithelial (HAE) model. In this system we again observed that both B.1.617.2 isolates had a significant replication advantage over B.1.1.7 (Fig. ). Finally, we infected primary three-dimensional airway organoids (Fig. ) with B.1.617.2 and B.1.1.7 virus isolates, noting a significant replication advantage for B.1.617.2 over B.1.1.7. These data clearly support the higher replication rate and therefore transmissibility of B.1.617.2 over B.1.1.7. Fig. 2 Delta variant live virus replication kinetics and spike-mediated infectivity. a - d, Live virus replication comparing B.1.1.7 with B....
B.1.617.2 vaccine breakthrough infection
We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs) started in early 2021 with the ChAdOx1 vaccine (Covishield). During the wave of infections in March and April, symptomatic SARS-CoV-2 was confirmed in 30 vaccinated staff members among a workforce of 3,800 at a single tertiary centre in Delhi. Genomic data from India and Delhi suggested B.1.1.7 dominance (Fig. and Extended Data Fig. ), with growth of B.1.617 during March 2021. Short-read sequencing of symptomatic non-fatal infections in the HCW outbreak revealed that the majority were B.1.617.2 with a range of other B lineage viruses (Fig. ). Phylogenetic analysis demonstrated a group of highly related, and in some cases, genetically indistinct sequences that were sampled within 1 or 2 days of each other (Fig....
B.1.617.2 vaccine breakthrough infection
Across the three centres, we noted that the median age and duration of infection of those infected with B.1.617.2 versus non-B.1.617.2 were similar (Extended Data Table ), with no evidence that B.1.617.2 was associated with higher risk of hospitalization (Extended Data Table ). Next we evaluated the effect of B.1.617.2 on vaccine effectiveness against symptomatic infection in the HCWs, compared with other lineages. We used multivariable logistic regression to estimate the odds ratio of testing positive with B.1.617.2 versus non-B.1.617.2 in vaccinated relative to unvaccinated individuals, adjusting for age, sex and hospital. The adjusted odds ratio for B.1.617.2 relative to non-B.1.617.2 was 5.45 (95% confidence interval 1.39-21.4, P = 0.018) for two vaccine doses (Extended Data Table ).
Vaccine effectiveness
To estimate vaccine effectiveness for the B.1.617.2 variant relative to non-B.1.617.2 variants, we adopted a recently described approach. This method is based on the premise that if the vaccine is equally effective against B.1.617.2 and non-B.1.617.2 variants, a similar proportion of cases with each variant would be expected in both vaccinated and unvaccinated cases. This approach overcomes the issue of higher background prevalence of one variant over the other. We determined the proportion of individuals with the B.1.617.2 variant relative to all other circulating variants by vaccination status. We then used logistic regression to estimate the odds ratio of testing positive with B.1.617.2 in vaccinated compared with unvaccinated individuals. The final regression model was adjusted for age as a continuous variable, and sex and hospital as categorical variables. Model sensitivity and...
Standardization of virus input by SYBR Green-based product-enhanced PCR assay
The reverse transcriptase (RT) activity of virus preparations was determined by quantitative PCR (qPCR) using a SYBR Green-based product-enhanced PCR assay as previously described. In brief, tenfold dilutions of virus supernatant were lysed in a 1:1 ratio in a 2 × lysis solution (made up of 40% glycerol (v/v), 0.25% Triton X-100 (v/v), 100 mM KCl, RNase inhibitor 0.8 U ml -1, Tris HCl 100 mM, buffered to pH 7.4) for 10 min at room temperature.
Viral isolate comparison between B.1.617.1 and B.1.617.2
Vero E6 TMPRSS2 cells (an African green monkey ( Chlorocebus sabaeus ) kidney cell line; JCRB1819) were maintained in DMEM (low glucose) (Wako, catalogue no. 041-29775) containing 10% FCS, G418 (1 mg ml -1; Nacalai Tesque, catalogue no. G8168-10ML) and 1% antibiotics (penicillin and streptomycin (P/S)).
Viral isolate comparison between B.1.617.1 and B.1.617.2
Calu-3 cells (a human lung epithelial cell line; ATCC HTB-55) were maintained in minimum essential medium Eagle (Sigma-Aldrich, catalogue no. M4655-500ML) containing 10% FCS and 1% PS.
Measurement outputs
What raw and processed outputs should exist?
We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs)...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
a Wilcoxon rank-sum test.
from paperScoring or quantification
Quantify the primary readouts for this experiment: We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase...; We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs)...; Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then...; Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were....
from paperStatistical comparison
a Wilcoxon rank-sum test. b Chi square test. c 111 of 112 available. d 11 of 112. e 2 of 20. f 63 of 112. g 12 of 20. † Vaccine status missing for 1 of 132. *Hospitalisati...; We first plotted the relative proportion of variants in new cases of SARS-CoV-2 in India since the start of 2021. Although B.1.617.1 emerged earlier, the Delta variant B.1.617.2...; We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChA...; We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase...
from paperReporting output
Report representative outputs alongside summary comparisons for We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase..., We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs)..., Three sets of fasta consensus sequences were obtained from three separate hospitals in Delhi, India. Initially, all sequences were concatenated into a multi-fasta file and then..., Phylogenies were inferred using maximum likelihood in IQTREE v2.1.4 (ref. ) using a GTR + R6 model with 1,000 rapid bootstraps. The inferred phylogenies were....
inferred from protocolStructured statistical methods
a Wilcoxon rank-sum test. b Chi square test. c 111 of 112 available. d 11 of 112. e 2 of 20. f 63 of 112. g 12 of 20. † Vaccine status missing for 1 of 132. *Hospitalisati...; We first plotted the relative proportion of variants in new cases of SARS-CoV-2 in India since the start of 2021. Although B.1.617.1 emerged earlier, the Delta variant B.1.617.2...; We used the same B.1.617.2 live virus isolate to test susceptibility to vaccine-elicited serum neutralizing antibodies in individuals following vaccination with two doses of ChA...; We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Extended Data Table 4 Relative recei1 vaccine effectiveness against B1.617.2 v non- B1.617.2: Upper Table: Odds ratios for detection of B.1.617.2 relative to non-B.1.617.2 in vaccinated compared to unvaccinated individuals in multi-variable logistic regression
We first infected a lung epithelial cell line, Calu-3, comparing B.1.1.7 and B.1.617.2 (Fig. ). We observed a replication advantage for B.1.617.2 (Fig. ), as well as an increase in released virions from cells (Fig. ). Next we tested B.1.1.7 against two separate isolates of B.1.617.2 in a human airway epithelial (HAE) model. In this system we again observed that both B.1.617.2 isolates had a significant replication advantage over B.1.1.7 (Fig. ). Finally, we infected primary three-dimensional airway organoids (Fig. ) with B.1.617.2 and B.1.1.7 virus isolates, noting a significant replication advantage for B.1.617.2 over B.1.1.7. These data clearly support the higher replication rate and therefore transmissibility of B.1.617.2 over B.1.1.7. Fig. 2 Delta variant live virus replication kinetics and spike-mediated infectivity. a - d, Live virus replication comparing B.1.1.7 with B.1.617.2. Calu-3 cells were infected with variants at an MOI of 0.1. a, Viral loads measured by qPCR in cell lysates. b, Viral protein levels in cell lysates. c, d, Live virus produced from infected Calu-3 cell supernatants was collected and used to infect pe...
We hypothesized that vaccine effectiveness against B.1.617.2 would be compromised relative to that against other circulating variants. Vaccination of health care workers (HCWs) started in early 2021 with the ChAdOx1 vaccine (Covishield). During the wave of infections in March and April, symptomatic SARS-CoV-2 was confirmed in 30 vaccinated staff members among a workforce of 3,800 at a single tertiary centre in Delhi. Genomic data from India and Delhi suggested B.1.1.7 dominance (Fig. and Extended Data Fig. ), with growth of B.1.617 during March 2021. Short-read sequencing of symptomatic non-fatal infections in the HCW outbreak revealed that the majority were B.1.617.2 with a range of other B lineage viruses (Fig. ). Phylogenetic analysis demonstrated a group of highly related, and in some cases, genetically indistinct sequences that were sampled within 1 or 2 days of each other (Fig. and Extended Data Fig. ). We next looked in greater detail at the vaccination history of affected individuals. Nearly all had received two doses at least 21 days previously. We obtained similar data on vaccine breakthrough infections in two other health facilities in Delhi with 1,100 and 4,000 HCW s...
Across the three centres, we noted that the median age and duration of infection of those infected with B.1.617.2 versus non-B.1.617.2 were similar (Extended Data Table ), with no evidence that B.1.617.2 was associated with higher risk of hospitalization (Extended Data Table ). Next we evaluated the effect of B.1.617.2 on vaccine effectiveness against symptomatic infection in the HCWs, compared with other lineages. We used multivariable logistic regression to estimate the odds ratio of testing positive with B.1.617.2 versus non-B.1.617.2 in vaccinated relative to unvaccinated individuals, adjusting for age, sex and hospital. The adjusted odds ratio for B.1.617.2 relative to non-B.1.617.2 was 5.45 (95% confidence interval 1.39-21.4, P = 0.018) for two vaccine doses (Extended Data Table ).
To estimate vaccine effectiveness for the B.1.617.2 variant relative to non-B.1.617.2 variants, we adopted a recently described approach. This method is based on the premise that if the vaccine is equally effective against B.1.617.2 and non-B.1.617.2 variants, a similar proportion of cases with each variant would be expected in both vaccinated and unvaccinated cases. This approach overcomes the issue of higher background prevalence of one variant over the other. We determined the proportion of individuals with the B.1.617.2 variant relative to all other circulating variants by vaccination status. We then used logistic regression to estimate the odds ratio of testing positive with B.1.617.2 in vaccinated compared with unvaccinated individuals. The final regression model was adjusted for age as a continuous variable, and sex and hospital as categorical variables. Model sensitivity and robustness to inclusion of these covariates was tested by an iterative process of sequentially adding the covariates to the model and examining the impact on the odd ratios and confidence intervals until the final model was constructed (Extended Data Table ). The R 2 measure, as proposed by McFadden...
The reverse transcriptase (RT) activity of virus preparations was determined by quantitative PCR (qPCR) using a SYBR Green-based product-enhanced PCR assay as previously described. In brief, tenfold dilutions of virus supernatant were lysed in a 1:1 ratio in a 2 × lysis solution (made up of 40% glycerol (v/v), 0.25% Triton X-100 (v/v), 100 mM KCl, RNase inhibitor 0.8 U ml -1, Tris HCl 100 mM, buffered to pH 7.4) for 10 min at room temperature.
Vero E6 TMPRSS2 cells (an African green monkey ( Chlorocebus sabaeus ) kidney cell line; JCRB1819) were maintained in DMEM (low glucose) (Wako, catalogue no. 041-29775) containing 10% FCS, G418 (1 mg ml -1; Nacalai Tesque, catalogue no. G8168-10ML) and 1% antibiotics (penicillin and streptomycin (P/S)).
Calu-3 cells (a human lung epithelial cell line; ATCC HTB-55) were maintained in minimum essential medium Eagle (Sigma-Aldrich, catalogue no. M4655-500ML) containing 10% FCS and 1% PS.
Machine-readable layer
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"text": "To estimate vaccine effectiveness for the B.1.617.2 variant relative to non-B.1.617.2 variants, we adopted a recently described approach. This method is based on the premise that if the vaccine is equally effective against B.1.617.2 and non-B.1.617.2 variants, a similar proportion of cases with each variant would be expected in both vaccinated and unvaccinated cases. This approach overcomes the issue of higher background prevalence of one variant over the other. We determined the proportion of individuals with the B.1.617.2 variant relative to all other circulating variants by vaccination status. We then used logistic regression to estimate the odds ratio of testing positive with B.1.617.2 in vaccinated compared with unvaccinated individuals. The final regression model was adjusted for age as a continuous variable, and sex and hospital as categorical variables. Model sensitivity and..."
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"text": "Vero E6 TMPRSS2 cells (an African green monkey ( Chlorocebus sabaeus ) kidney cell line; JCRB1819) were maintained in DMEM (low glucose) (Wako, catalogue no. 041-29775) containing 10% FCS, G418 (1 mg ml -1; Nacalai Tesque, catalogue no. G8168-10ML) and 1% antibiotics (penicillin and streptomycin (P/S))."
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