Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity methods
Aim. Evidence-backed execution summary for Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity methods from Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.
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human
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Main
reagent used in the protocol.
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- The Omicron variant of SARS-CoV-2, which was first detected in South Africa, carries over 30 mutations in its spike protein and has now spread internationally at a fast pace. More than 20 substitutions exist in the N-terminal domain and receptor-binding domain (RBD). The Omicron variant also contains six unique mut...
Omicron spike-binding affinity to ACE2
reagent used in the protocol.
- Use
- The Omicron variant has 15 amino acid mutations in the RBD (Supplementary Figs. and ). To understand the impact of these substitutions on receptor engagement, we determined the kinetics and affinity of monomeric human ACE2 binding to immobilized Omicron, Wuhan-Hu-1 and Delta RBDs using biolayer interferometry. We ob...
The sensitivity of Omicron to therapeutics
reagent used in the protocol.
- Use
- Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care antiviral treatment for moderate to severe COVID-19 includes the use of the monoclonal antibody combination REGN10933 (casivirimab) and REG...
Omicron and polyclonal antibodies
reagent used in the protocol.
- Use
- An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike proteins and generated pseudotyped virus (PV) particles by co-transfecting the spike expression plasmids with a lentiviral gag-pol-expressi...
Omicron and polyclonal antibodies
reagent used in the protocol.
- Use
- To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. )...
Omicron replication and spike cleavage
reagent used in the protocol.
- Use
- We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified u...
Omicron entry is impaired in TMPRSS2 + cells
reagent used in the protocol.
- Use
- A change in the use of TMPRSS2 for entry would be predicted to alter the entry pathway of the virus (Fig. ). To test the hypothesis that the entry route preference by Omicron spike is changed, we used inhibitors of proteases specific to either the endocytic pathway (E64d blockade of cathepsins) or the plasma membran...
Impaired cell-cell fusion by Omicron S
reagent used in the protocol.
- Use
- Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrate...
Supplementary information
Supplementary Information Uncropped western blots for Fig. 2; the FACS gating strategy for Extended Data Fig.8; and the uncropped western blots for Extended Data Fig. 6.
- Use
- Supplementary Information Uncropped western blots for Fig. 2; the FACS gating strategy for Extended Data Fig.8; and the uncropped western blots for Extended Data Fig. 6.
Omicron and polyclonal antibodies
To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. )...
- Use
- To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. )...
Omicron replication and spike cleavage
We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified u...
- Use
- We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified u...
Omicron entry is impaired in TMPRSS2 + cells
We tested spike-mediated viral entry of the wild-type (WT) Wuhan-Hu-1 D614G, Delta and Omicron spike proteins (Fig. ) using the PV system. We first probed PV virions for spike protein and, after western blot analysis, noted a reduced incorporation of Omicron spike into virions compared with the incorporation of Delt...
- Use
- We tested spike-mediated viral entry of the wild-type (WT) Wuhan-Hu-1 D614G, Delta and Omicron spike proteins (Fig. ) using the PV system. We first probed PV virions for spike protein and, after western blot analysis, noted a reduced incorporation of Omicron spike into virions compared with the incorporation of Delt...
Omicron entry is impaired in TMPRSS2 + cells
To experimentally demonstrate differential usage of TMPRSS2 as a cofactor for virus entry by Omicron, we directly compared the efficiency of viral entry between 293T-A2ΔT2 and 293T-A2T2 cells. Enhanced infectivity was observed for both the WT and Delta when TMPRSS2 was overexpressed, suggesting that TMPRSS2 is...
- Use
- To experimentally demonstrate differential usage of TMPRSS2 as a cofactor for virus entry by Omicron, we directly compared the efficiency of viral entry between 293T-A2ΔT2 and 293T-A2T2 cells. Enhanced infectivity was observed for both the WT and Delta when TMPRSS2 was overexpressed, suggesting that TMPRSS2 is...
Omicron entry is impaired in TMPRSS2 + cells
Having established that TMPRSS2 modulates entry mediated by Delta to a greater extent than for Omicron BA.1 spike, we sought to understand the distribution of TMPRSS2 and ACE2 expression in human respiratory cells. We used single-nucleus RNA-sequencing (RNA-seq) data from five locations in the human lungs. The comp...
- Use
- Having established that TMPRSS2 modulates entry mediated by Delta to a greater extent than for Omicron BA.1 spike, we sought to understand the distribution of TMPRSS2 and ACE2 expression in human respiratory cells. We used single-nucleus RNA-sequencing (RNA-seq) data from five locations in the human lungs. The comp...
Impaired cell-cell fusion by Omicron S
Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrate...
- Use
- Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrate...
Impaired cell-cell fusion by Omicron S
We performed flow cytometry to verify that spike was expressed at the cell surface (Fig. ). We proceeded to transfect spike-bearing plasmids into HEK293T cells expressing GFP1-10 and Vero E6 cells stably expressing GFP11, such that the GFP signal could be detected after cell-cell fusion and measured over time....
- Use
- We performed flow cytometry to verify that spike was expressed at the cell surface (Fig. ). We proceeded to transfect spike-bearing plasmids into HEK293T cells expressing GFP1-10 and Vero E6 cells stably expressing GFP11, such that the GFP signal could be detected after cell-cell fusion and measured over time....
The sensitivity of Omicron to therapeutics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care antiviral treatment for moderate to severe COVID-19 includes the use of the monoclonal antibody combination REGN10933 (casivirimab) and REG...
Neutralization assay for monoclonal antibodies with live virus
Software used for acquisition, scoring, statistics, or reporting.
- Use
- One day before infection, VeroE6/TMPRSS2 (10,000 cells) were seeded into a 96-well plate. The monoclonal antibodies (casirivimab, imdevimab or casirivimab/imdevimab) and the heat-inactivated human sera were serially diluted with DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. The diluted a...
Antiviral drug assay with live virus
Software used for acquisition, scoring, statistics, or reporting.
- Use
- One day before infection, HOS-ACE2-TMPRSS2 cells (10,000 cells) were seeded into a 96-well plate. The cells were infected with SARS-CoV-2 (100 TCID 50 ) Omicron (EPI_ISL_7418017) or Delta (EPI_ISL_2378732) isolates at 37 °C for 1 h. The cells were next washed with DMEM and cultured in DMEM supplemen...
PV SG-PERT and infectivity
Software used for acquisition, scoring, statistics, or reporting.
- Use
- PVs were used to transduce different target cell lines and 3D organoid cultures. After 48 or 72 h after transduction, the cells were lysed with direct addition of Bright Glo (Promega). The raw readings were then normalized to the SG-PERT and plotted using GraphPad Prism.
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The sensitivity of Omicron to therapeutics
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care antiviral treatment for moderate to severe COVID-19 includes the use of the monoclonal antibody combination REGN10933 (casivirimab) and REGN10897 (imdevimab). The K417N, E484A, S477N and Q493R mutations preclude REGN10933 binding, whereas G446S clashes with REGN10987, consistent with the decreased binding to the Omicron RBD and S trimer and the loss of neutralization previously descibed,. We next tested serial dilutions of component monoclonal antibodies, both individually and in combination, against Delta and Omicron live viruses in tissue culture (Fig. ). Although the Delta variant was effectively neutralized by casirivimab, imdevimab was only partially effective, consistent with previous data. In co...
Omicron and polyclonal antibodies
An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike proteins and generated pseudotyped virus (PV) particles by co-transfecting the spike expression plasmids with a lentiviral gag-pol-expressing plasmid and a lentiviral transduction plasmid encoding the luciferase gene,. We obtained longitudinal serum samples from 40 individuals who were vaccinated with either the BNT162b2 or ChAdOx-1 vaccine and performed serum titrations before mixing sera with our reporter PV particles. The participants had a median age of around 70 years and prospective serum samples were taken as follows: one month after dose two, six months after dose two and one month after dose three (Extended Data Table ). We observed at least tenfold loss of neutralization against Omicron after the s...
Omicron and polyclonal antibodies
To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. ). These live viruses were also used to assess the neutralization of the Omicron variant by sera derived from unvaccinated individuals who were previously infected with the early Wuhan-Hu-1 virus or the Delta variant. As expected, vaccine sera had significantly impaired activity against Omicron compared with Delta (Extended Data Fig. ). We also tested mRNA-1273 vaccine-elicited sera, which showed a similar reduction in neutralization to BNT162b2. However, coronavac sera showed little neutralization against Delta and 0 out of 9 participants had detectable neutralization again...
Omicron replication and spike cleavage
We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified using quantitative PCR (qPCR) analysis of the E gene and 50% tissue culture infectious dose (TCID 50 ). We observed similar replication kinetics for Omicron and Delta in the hNECs (Fig. ). We next infected Calu-3 lung cells (which are known to express endogenous TMPRSS2 ) with live isolates, and observed significantly greater viral replication for Delta than Omicron (Fig. ), manifesting as early as 24 h after infection. We also infected Caco-2 cells, a colon cancer cell line (known to express high levels of endogenous TMPRSS2 ) and, as for Calu-3, we found 1 log...
Impaired cell-cell fusion by Omicron S
The ability of viral membrane glycoproteins to induce cell-cell fusion and syncytium formation is well established -, providing an additional route for SARS-CoV-2 dissemination that may facilitate evasion of neutralizing antibodies. The role of syncytium formation in viral replication and the pathogenesis of severe COVID-19 has been reported and may be a druggable process to treat COVID-19 pathology,. SARS-CoV-2-mediated cell-cell fusion requires the PBCS and spike cleavage at S1/S2, and the process is known to be accelerated by the presence of TMPRSS2 (ref. ).
Impaired cell-cell fusion by Omicron S
Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrated CMK (a furin inhibitor) into donor cell medium before transfection with spike-expressing plasmids, observing dose-dependent inhibition of fusion (Extended Data Fig. ). As a further control to demonstrate the need for ACE2 engagement by expressed spike, we tested the ability of convalescent serum containing neutralizing antibodies to block cell-cell fusion. Indeed, serum blocked syncytia formation in a dose-dependent manner (Extended Data Fig. ). Fig. 4 SARS-CoV-2 Omicron variant spike shows impaired cell-cell fusion activity and smaller infection foci generate...
Impaired cell-cell fusion by Omicron S
We predicted that poor fusion would impair cell-cell spread of Omicron, and analysed infection focus size using live virus infection of H1299 cells, in which we have shown similar entry efficiency for Omicron and Delta spike PV (Fig. ). Infection foci in spreading virus infections occur because of localized (cell-to-cell) infection, probably facilitated by syncytium formation. The infection was followed by overlay of semi-solid medium (carboxymethylcellulose) to inhibit cell-free infection, thereby favouring cell-cell infection. Infected cells were stained with an anti-S antibodies, and local spread was characterized by the staining of foci visible to the naked eye (Fig. ). Omicron infection resulted in slightly higher numbers of foci (Fig. ), but each infection focus was substantially smaller in size relative to those formed by Delta infection (Fig. ). For a more detailed...
Flow cytometry analysis of spike protein surface expression
ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 10 6 cells per ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After incubation for 1 min at room temperature, transfection mixes were added to the cell suspensions. Next, the cells were incubated at 37 °C under 8% CO 2 with an orbital shaking speed of 120 rpm for the next 48 h.
Measurement outputs
What raw and processed outputs should exist?
The Omicron variant has 15 amino acid mutations in the RBD (Supplementary Figs. and ). To understand the impact of these substitutions on receptor engagement, we determined the...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care anti...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the a...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The Omicron variant has 15 amino acid mutations in the RBD (Supplementary Figs. and ). To understand the impact of these substitutions on receptor engagement, we determined the...; Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care anti...; An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike...; We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the a....
from paperStatistical comparison
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care anti...; We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the a...; We tested spike-mediated viral entry of the wild-type (WT) Wuhan-Hu-1 D614G, Delta and Omicron spike proteins (Fig. ) using the PV system. We first probed PV virions for spike p...; Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monito...
from paperReporting output
Report representative outputs alongside summary comparisons for The Omicron variant has 15 amino acid mutations in the RBD (Supplementary Figs. and ). To understand the impact of these substitutions on receptor engagement, we determined the..., Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care anti..., An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike..., We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the a....
inferred from protocolStructured statistical methods
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care anti...; We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the a...; We tested spike-mediated viral entry of the wild-type (WT) Wuhan-Hu-1 D614G, Delta and Omicron spike proteins (Fig. ) using the PV system. We first probed PV virions for spike p...; Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monito...
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Evidence quotes (8)
Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care antiviral treatment for moderate to severe COVID-19 includes the use of the monoclonal antibody combination REGN10933 (casivirimab) and REGN10897 (imdevimab). The K417N, E484A, S477N and Q493R mutations preclude REGN10933 binding, whereas G446S clashes with REGN10987, consistent with the decreased binding to the Omicron RBD and S trimer and the loss of neutralization previously descibed,. We next tested serial dilutions of component monoclonal antibodies, both individually and in combination, against Delta and Omicron live viruses in tissue culture (Fig. ). Although the Delta variant was effectively neutralized by casirivimab, imdevimab was only partially effective, consistent with previous data. In combination, these monoclonal antibodies were highly potent against Delta. However, there was a complete loss of neutralizing activity against Omicron by these monoclonal antibodies alone or in combination (Fig. ). Given these results, we next tested the direct-acting antivirals remdesivir and the act...
An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike proteins and generated pseudotyped virus (PV) particles by co-transfecting the spike expression plasmids with a lentiviral gag-pol-expressing plasmid and a lentiviral transduction plasmid encoding the luciferase gene,. We obtained longitudinal serum samples from 40 individuals who were vaccinated with either the BNT162b2 or ChAdOx-1 vaccine and performed serum titrations before mixing sera with our reporter PV particles. The participants had a median age of around 70 years and prospective serum samples were taken as follows: one month after dose two, six months after dose two and one month after dose three (Extended Data Table ). We observed at least tenfold loss of neutralization against Omicron after the second dose compared with Delta (Fig. and Extended Data Fig. ). Indeed, neutralization of Omicron was not detectable for the majority of individuals who had received two doses of ChAdOx-1. We also observed waning over time after the second dose for both vaccines (Fig. ). Both groups received a booste...
To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. ). These live viruses were also used to assess the neutralization of the Omicron variant by sera derived from unvaccinated individuals who were previously infected with the early Wuhan-Hu-1 virus or the Delta variant. As expected, vaccine sera had significantly impaired activity against Omicron compared with Delta (Extended Data Fig. ). We also tested mRNA-1273 vaccine-elicited sera, which showed a similar reduction in neutralization to BNT162b2. However, coronavac sera showed little neutralization against Delta and 0 out of 9 participants had detectable neutralization against Omicron. Sera from Delta infections appeared to have lower cross-neutralization compared with those from the early pandemic period when Wuhan-Hu-1 D614G was dominant (Extended Data Fig. ).
We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified using quantitative PCR (qPCR) analysis of the E gene and 50% tissue culture infectious dose (TCID 50 ). We observed similar replication kinetics for Omicron and Delta in the hNECs (Fig. ). We next infected Calu-3 lung cells (which are known to express endogenous TMPRSS2 ) with live isolates, and observed significantly greater viral replication for Delta than Omicron (Fig. ), manifesting as early as 24 h after infection. We also infected Caco-2 cells, a colon cancer cell line (known to express high levels of endogenous TMPRSS2 ) and, as for Calu-3, we found 1 log greater Delta RNA and higher infectious virus (TCID 50 ) in the supernatants compared with those observed for Omicron. Finally, we tested a cell line-HeLa, overexpressing ACE2 and TMPRSS2 -and obtained similar results to those of the Calu-3 and Caco-2 cells. Fig. 2 SARS-CoV-2 Omicron and...
The ability of viral membrane glycoproteins to induce cell-cell fusion and syncytium formation is well established -, providing an additional route for SARS-CoV-2 dissemination that may facilitate evasion of neutralizing antibodies. The role of syncytium formation in viral replication and the pathogenesis of severe COVID-19 has been reported and may be a druggable process to treat COVID-19 pathology,. SARS-CoV-2-mediated cell-cell fusion requires the PBCS and spike cleavage at S1/S2, and the process is known to be accelerated by the presence of TMPRSS2 (ref. ).
Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrated CMK (a furin inhibitor) into donor cell medium before transfection with spike-expressing plasmids, observing dose-dependent inhibition of fusion (Extended Data Fig. ). As a further control to demonstrate the need for ACE2 engagement by expressed spike, we tested the ability of convalescent serum containing neutralizing antibodies to block cell-cell fusion. Indeed, serum blocked syncytia formation in a dose-dependent manner (Extended Data Fig. ). Fig. 4 SARS-CoV-2 Omicron variant spike shows impaired cell-cell fusion activity and smaller infection foci generated by live virus. a, Schematic of the cell-cell fusion assay. The schematic was created using BioRender.com. b, Spike expression at the cell surface as determined by flow cytometry, showing the distribution of fluorescence intensity. The percentage of spike-positive cells is indicated....
We predicted that poor fusion would impair cell-cell spread of Omicron, and analysed infection focus size using live virus infection of H1299 cells, in which we have shown similar entry efficiency for Omicron and Delta spike PV (Fig. ). Infection foci in spreading virus infections occur because of localized (cell-to-cell) infection, probably facilitated by syncytium formation. The infection was followed by overlay of semi-solid medium (carboxymethylcellulose) to inhibit cell-free infection, thereby favouring cell-cell infection. Infected cells were stained with an anti-S antibodies, and local spread was characterized by the staining of foci visible to the naked eye (Fig. ). Omicron infection resulted in slightly higher numbers of foci (Fig. ), but each infection focus was substantially smaller in size relative to those formed by Delta infection (Fig. ). For a more detailed view of the foci, we imaged at a higher magnification using a microscope objective, and used cells that were imaged 2 h after infection with ancestral virus-when relatively little viral transmission is expected to be completed-as an approximate measure of the size of a single cell...
ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 10 6 cells per ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After incubation for 1 min at room temperature, transfection mixes were added to the cell suspensions. Next, the cells were incubated at 37 °C under 8% CO 2 with an orbital shaking speed of 120 rpm for the next 48 h.
Machine-readable layer
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"name": "The sensitivity of Omicron to therapeutics",
"text": "Omicron was predicted to have broad resistance to neutralizing antibodies based on mutations in the class I-IV antigenic regions in its RBD. Current standard of care antiviral treatment for moderate to severe COVID-19 includes the use of the monoclonal antibody combination REGN10933 (casivirimab) and REGN10897 (imdevimab). The K417N, E484A, S477N and Q493R mutations preclude REGN10933 binding, whereas G446S clashes with REGN10987, consistent with the decreased binding to the Omicron RBD and S trimer and the loss of neutralization previously descibed,. We next tested serial dilutions of component monoclonal antibodies, both individually and in combination, against Delta and Omicron live viruses in tissue culture (Fig. ). Although the Delta variant was effectively neutralized by casirivimab, imdevimab was only partially effective, consistent with previous data. In co..."
},
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"name": "Omicron and polyclonal antibodies",
"text": "An important question is whether vaccine-elicited antibodies are able to neutralize Omicron. We synthesized codon-optimized spike expression plasmids for Omicron and Delta spike proteins and generated pseudotyped virus (PV) particles by co-transfecting the spike expression plasmids with a lentiviral gag-pol-expressing plasmid and a lentiviral transduction plasmid encoding the luciferase gene,. We obtained longitudinal serum samples from 40 individuals who were vaccinated with either the BNT162b2 or ChAdOx-1 vaccine and performed serum titrations before mixing sera with our reporter PV particles. The participants had a median age of around 70 years and prospective serum samples were taken as follows: one month after dose two, six months after dose two and one month after dose three (Extended Data Table ). We observed at least tenfold loss of neutralization against Omicron after the s..."
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{
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"text": "To confirm the loss of neutralizing activity against Omicron after the second dose, we next used a live virus experimental system to compare Delta and Omicron variants against sera taken four weeks after the second dose of BNT162b2, and obtained similar results to those obtained in the PV assay (Extended Data Fig. ). These live viruses were also used to assess the neutralization of the Omicron variant by sera derived from unvaccinated individuals who were previously infected with the early Wuhan-Hu-1 virus or the Delta variant. As expected, vaccine sera had significantly impaired activity against Omicron compared with Delta (Extended Data Fig. ). We also tested mRNA-1273 vaccine-elicited sera, which showed a similar reduction in neutralization to BNT162b2. However, coronavac sera showed little neutralization against Delta and 0 out of 9 participants had detectable neutralization again..."
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"text": "We infected primary human nasal epithelial 3D cultures (hNECs) with an air-liquid interface (Fig. ). Infection with live SARS-CoV-2 Omicron or Delta was conducted at the apical surface with equal amounts of input virus and virus collected from the apical surface at 24 and 48 h, and virus was quantified using quantitative PCR (qPCR) analysis of the E gene and 50% tissue culture infectious dose (TCID 50 ). We observed similar replication kinetics for Omicron and Delta in the hNECs (Fig. ). We next infected Calu-3 lung cells (which are known to express endogenous TMPRSS2 ) with live isolates, and observed significantly greater viral replication for Delta than Omicron (Fig. ), manifesting as early as 24 h after infection. We also infected Caco-2 cells, a colon cancer cell line (known to express high levels of endogenous TMPRSS2 ) and, as for Calu-3, we found 1 log..."
},
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"name": "Impaired cell-cell fusion by Omicron S",
"text": "The ability of viral membrane glycoproteins to induce cell-cell fusion and syncytium formation is well established -, providing an additional route for SARS-CoV-2 dissemination that may facilitate evasion of neutralizing antibodies. The role of syncytium formation in viral replication and the pathogenesis of severe COVID-19 has been reported and may be a druggable process to treat COVID-19 pathology,. SARS-CoV-2-mediated cell-cell fusion requires the PBCS and spike cleavage at S1/S2, and the process is known to be accelerated by the presence of TMPRSS2 (ref. )."
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"name": "Impaired cell-cell fusion by Omicron S",
"text": "Given the requirement of TMPRSS2 for optimal cell-cell fusion, we hypothesized that Omicron may be impaired in mediating this process. We used a split GFP system to monitor cell-cell fusion in real time (Fig. ). As a control to show that spike cleavage is needed for fusion in our assay system, we titrated CMK (a furin inhibitor) into donor cell medium before transfection with spike-expressing plasmids, observing dose-dependent inhibition of fusion (Extended Data Fig. ). As a further control to demonstrate the need for ACE2 engagement by expressed spike, we tested the ability of convalescent serum containing neutralizing antibodies to block cell-cell fusion. Indeed, serum blocked syncytia formation in a dose-dependent manner (Extended Data Fig. ). Fig. 4 SARS-CoV-2 Omicron variant spike shows impaired cell-cell fusion activity and smaller infection foci generate..."
},
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"name": "Impaired cell-cell fusion by Omicron S",
"text": "We predicted that poor fusion would impair cell-cell spread of Omicron, and analysed infection focus size using live virus infection of H1299 cells, in which we have shown similar entry efficiency for Omicron and Delta spike PV (Fig. ). Infection foci in spreading virus infections occur because of localized (cell-to-cell) infection, probably facilitated by syncytium formation. The infection was followed by overlay of semi-solid medium (carboxymethylcellulose) to inhibit cell-free infection, thereby favouring cell-cell infection. Infected cells were stained with an anti-S antibodies, and local spread was characterized by the staining of foci visible to the naked eye (Fig. ). Omicron infection resulted in slightly higher numbers of foci (Fig. ), but each infection focus was substantially smaller in size relative to those formed by Delta infection (Fig. ). For a more detailed..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Flow cytometry analysis of spike protein surface expression",
"text": "ExpiCHO cells were seeded into 50 ml Mini Bioreactor Centrifuge Tube (Corning) at 6 × 10 6 cells per ml in 5 ml ExpiCHO Expression medium (Life technology). Plasmids encoding SARS-CoV-2 Wuhan, Alpha (B.1.1.7), Delta (B.1.617.2) or Omicron (B.1.1.529) spike protein (5 mg) were diluted in OptiPRO SFM (Life Technology) and mixed with Expifectamine CHO Reagent (Life Technology). After incubation for 1 min at room temperature, transfection mixes were added to the cell suspensions. Next, the cells were incubated at 37 °C under 8% CO 2 with an orbital shaking speed of 120 rpm for the next 48 h."
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