hek293-ace2 cell line  (Codex Biosolutions)

Journal: Journal of Virology

Article Title: Targeting envelope lipids with 2,6‑di‑ O ‑methyl‑3‑acetyl‑β‑cyclodextrin impairs infectivity of SARS‑CoV‑2 and Japanese encephalitis virus

doi: 10.1128/jvi.01357-25

Figure Lengend Snippet: DMA-β-CyD acts directly on SARS-CoV-2 and reduces its infectivity titer. DMA-β-CyD was directly exposed to different SARS-CoV-2 variants for ( A ) 10 min and ( B ) 180 min. Viral infectivity titers were expressed in TCID 50 /mL, and significant differences (Student’s t -test) are indicated in the figures. Mean values from at least three independent experiments are shown, and error bars indicate SD. ( C ) Competitive inhibition experiment with DMA-β-CyD. Recombinant ACE2 was used, and DMA-β-CyD was pre-incubated before the loading of the recombinant spike protein. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Dunnett’s test. n.s. indicates not significant. The mean values of at least three independent experiments are shown. The error bars denote SD.

Article Snippet: To determine whether DMA-β-CyD inhibits the binding of the SARS-CoV-2 spike protein to ACE2, an ACE2:SARS-CoV-2 Spike S1 Inhibitor Screening Assay Kit (BPS Bioscience, Inc.) was used.

Techniques: Infection, Inhibition, Recombinant, Incubation

Journal: Journal of Virology

Article Title: Targeting envelope lipids with 2,6‑di‑ O ‑methyl‑3‑acetyl‑β‑cyclodextrin impairs infectivity of SARS‑CoV‑2 and Japanese encephalitis virus

doi: 10.1128/jvi.01357-25

Figure Lengend Snippet: DMA-β-CyD prolongs the virus-induced reduction in clotting time in human serum by masking aminophospholipids in the viral lipid envelope. ( A ) The Wuhan, Beta, and Delta variants prepared in Vero E6/TMPRSS2 cells were treated with DMA-β-CyD for the indicated times, followed by a clotting assay. ( B ) The Wuhan variant, prepared in A549 cells overexpressing ACE2, was treated with DMA-β-CyD for the indicated times, followed by a clotting assay. ( C ) To examine the significance of the molecular encapsulation activity of DMA-β-CyD, pretreatment with 10 mM DMAβCyD with equimolar (10 mM) 1adamantanecarboxylic acid was performed to assess its effect on the virus-induced reduction in clotting time. ( D ) The Wuhan variant, prepared in Vero E6/TMPRSS2 cells, was treated with DM-β-CyD for the indicated times, followed by a clotting assay. P values were determined using one-way ANOVA (Dunnett’s test). * indicates a statistically significant difference in clotting time between groups untreated and treated with DMA-β-CyD or DM-β-CyD ( P < 0.05). Data represent the means of at least three independent experiments, and error bars indicate SD.

Article Snippet: To determine whether DMA-β-CyD inhibits the binding of the SARS-CoV-2 spike protein to ACE2, an ACE2:SARS-CoV-2 Spike S1 Inhibitor Screening Assay Kit (BPS Bioscience, Inc.) was used.

Techniques: Virus, Coagulation, Variant Assay, Encapsulation, Activity Assay

Journal: Journal of Virology

Article Title: Targeting envelope lipids with 2,6‑di‑ O ‑methyl‑3‑acetyl‑β‑cyclodextrin impairs infectivity of SARS‑CoV‑2 and Japanese encephalitis virus

doi: 10.1128/jvi.01357-25

Figure Lengend Snippet: Inhibition of viral entry by DMA-β-CyD and evaluation of viral particle stability. ( A ) DMA-β-CyD was preincubated with the SARS-CoV-2 Wuhan variant, which was prepared in Vero E6/TMPRSS2 cells. Free DMA-β-CyD was removed by gel filtration, and the resulting viral particles were then used to infect cells. Four hours after infection, total RNA was extracted from Vero E6/TMPRSS2 cells, and gRNA was detected by RT-PCR. P values were determined by one-way ANOVA using Dunnett’s test (** P < 0.01). N.D. indicates that the SARS-CoV-2 phenotypic RNA (gRNA) was not detected. Data represent the means of at least three independent experiments, and error bars indicate SD. ( B ) DMA-β-CyD was preincubated with the SARS-CoV-2 Wuhan variant, which was prepared in A549 cells overexpressing ACE2. The efficiency of viral entry was similarly examined. Western blot analysis results, shown in the bar graph inset, confirm ACE2 expression in A549 cells. ( C ) The SARS-CoV-2 Wuhan variant, prepared in Vero E6/TMPRSS2 cells, was treated with anti-SARS-CoV-2 patient serum or DMA-β-CyD for 180 min, followed by virus purification by ultracentrifugation and western blot analysis using patient antiserum. ( D ) Evaluation of virus particle stability. (Left panel) The SARS-CoV-2 Wuhan variant, prepared in Vero E6/TMPRSS2 cells, was treated with an anti-S2 antibody and then subjected to gel filtration. Gel filtration was also performed using mouse IgG and IgM as molecular weight markers. The resulting flow-through fraction was analyzed by western blot analysis. (Right panel) The SARS-CoV-2 Wuhan variant, prepared in Vero E6/TMPRSS2 cells, was treated with DMA-β-CyD or anti-SARS-CoV-2 serum. The virus was purified by gel filtration, captured on a plate coated with anti-RBD antibodies, and the number of plate-bound viral particles was quantified by RT-qPCR. P values were determined by one-way ANOVA using Dunnett’s test (** P < 0.01). N.D. indicates that no detectable SARS-CoV-2 gRNA was found. Data represent the means of at least three independent experiments, and error bars indicate SD.

Article Snippet: To determine whether DMA-β-CyD inhibits the binding of the SARS-CoV-2 spike protein to ACE2, an ACE2:SARS-CoV-2 Spike S1 Inhibitor Screening Assay Kit (BPS Bioscience, Inc.) was used.

Techniques: Inhibition, Variant Assay, Filtration, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Virus, Purification, Molecular Weight, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: S6P mutation in Delta and Omicron variant spike protein significantly enhances the efficacy of mRNA COVID-19 vaccines

doi: 10.3389/fimmu.2024.1495561

Figure Lengend Snippet: In vitro expression of mRNA encoding Spike protein with 2P or 6P mutations in various strains. (A) HEK293T cells were transfected with mRNA encoding different forms of the SARS-CoV-2 S protein. After 48 hours, cell lysates were analyzed by Western Blot using an anti-SARS-CoV-2 S2-specific antibody. (B–D) the cells underwent flow cytometry to measure surface expression, with staining performed using a Flag-tagged ACE2 receptor and the anti-S antibody clone H4 (B) The mean fluorescence intensity (MFI) was calculated to assess the cell surface expression levels, using the anti-Flag antibody (C) and the anti-S antibody clone H4 (D) .

Article Snippet: The SARS-CoV-2-PP was tested for the quality control with HEK293-ACE2 cell line (created at Codex BioSolutions).

Techniques: In Vitro, Expressing, Transfection, Western Blot, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: S6P mutation in Delta and Omicron variant spike protein significantly enhances the efficacy of mRNA COVID-19 vaccines

doi: 10.3389/fimmu.2024.1495561

Figure Lengend Snippet: Protective efficacy of RV-1730 against SARS-CoV-2/human/ITA/INMI1/2020 in a K18-hACE2 mouse model of lethal infection. K18-ACE2 mice (n = 10 per group) were immunized with 3 doses of 1, 5, and 10 µg RV-1730 per mouse and challenged with wild type SARS-Cov-2 virus as described in “Methods” section. Mice in Group 1 were administered PBS, Group 2 mice were immunized with mRNA RV-1730 1 µg/animal, Group 3 mice were immunized with mRNA RV-1730 5 µg/animal, Group 4 mice were immunized with mRNA RV-1730 10 µg/animal before viral challenge. (A) Kaplan Meier curve of survival rate for mice challenged with SARS-CoV-2/human/ITA/INMI1/2020 virus. (B) clinical severity score curve in immunized and non-immunized mice after challenge with SARS-CoV-2 virus. (C) Plaque reduction neutralizing test of mouse serum against SARS-CoV-2/human/ITA/INMI1/2020. PRNT 50 of post-prime immunization mouse serum collected on day 13 and day 27. Symbols and horizontal lines represent individual titers of each sample and mean titers of each group, respectively. Serum titers were expressed as reciprocals Log 2 dilution. ****P<0.0001 compared to PBS (one-way ANOVA with Dunnett’s test).

Article Snippet: The SARS-CoV-2-PP was tested for the quality control with HEK293-ACE2 cell line (created at Codex BioSolutions).

Techniques: Infection, Virus

Journal: Nature Communications

Article Title: De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

doi: 10.1038/s41467-024-53105-8

Figure Lengend Snippet: A Separation of the polyclonal antibody on a native gel. Initially, the enriched polyclonal antibody was digested with IdeS, and the resulting sample was fractionated into 12 fractions and further digested with trypsin. The separation was carried out once with IdeS and once without IdeS, respectively. Separation without Ides resulted in fewer fractions (see Source Data file). B Quantitative profiling of various peptide “contigs” across those 12 fractions, where contigs were grouped based on their similarity to assemble chains and pair heavy-light (HL) chains (more information on the contig sequence can be found in SI Table ). C top: shows an example of the sequence coverage resulting from EThcD analysis of trypsin and pepsin digestion. Peptides resulting from pepsin digestion were labeled with Arginine methyl ester. Bottom: displays EThcD spectra acquired to resolve specific Isoleucine/Leucine assignments; in that case, the generation of w-ions under EThcD fragmentation allows the assignment of w8 to an isoleucine. Additionally, in this case, a positive charge (i.e., Methyl Arginine as a y1 ion) was added to the C-terminal end of the non-tryptic peptide to facilitate the C-terminal fragment to be detectable in MS/MS mode. D Expressed candidates and their affinities in ELISA Assay, Angiotensin-converting enzyme 2, ACE2 competition binding assay and in vitro neutralization assay (Using either the Receptor-Binding Domain, RBD or the protein S1). Source data are provided as a Source Data file.

Article Snippet: For the in vitro, cell-based neutralization assay, HEK293/Human ACE2 Stable Cell Line (Acro Biosystems) was cultured in complete DMEM medium (Shanghai BasalMedia Technologies) supplemented with 10% fetal bovine serum (VivaCell) at 37 °C with 5% CO2.

Techniques: Sequencing, Labeling, Tandem Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Binding Assay, In Vitro, Neutralization

Journal: Nature Communications

Article Title: De novo protein sequencing of antibodies for identification of neutralizing antibodies in human plasma post SARS-CoV-2 vaccination

doi: 10.1038/s41467-024-53105-8

Figure Lengend Snippet: The Materials and Methods section describes how the various ELISA procedures were performed. A Conventional ELISA assay comparing recombinant antibodies with the natural polyclonal antibody PD124 affinity enriched in this study as a benchmark measure. The red dashed arrows are the IC50 for the best recombinant antibody (R2 at approx. 3 nM) versus the natural polyclonal antibody (PD124 at approx. 4 nM). Nonbinding recombinant antibodies (r3, r7, r8, r9, r10) were eliminated from further comparisons. Due to its restricted availability, the natural polyclonal antibody PD124 was excluded from other investigations. Figure 4B illustrates an ACE2 competitive binding assay conducted on the recombinant forms found in ( A ), utilizing the RBD domain. C ACE2 competitive binding assay that targets the S1 protein as the bait in the test. For A - C , although these ELISA assays focus on different aspects, they all utilized the horseradish peroxidase assay with TMB (3,3′,5,5′ tetramethylbenzidine, TMB, resulting in the measurement outcomes at 450 nm across the three different assays. D In vitro neutralization test that utilizes SARS-CoV-2-Spike/Flu-GFP pseudovirus to neutralize a positive standard S1N-M122. The assay measures the ratios of Absolute IC55. The positive control in B , C is AS35, an Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1, while in ( D ), it is the S1N-M122, a known anti-Spike RBD Neutralizing Antibody, Chimeric mAb, Human IgG1 (AM122), both sourced from Acro Biosystems. Source data are provided as a Source Data file.

Article Snippet: For the in vitro, cell-based neutralization assay, HEK293/Human ACE2 Stable Cell Line (Acro Biosystems) was cultured in complete DMEM medium (Shanghai BasalMedia Technologies) supplemented with 10% fetal bovine serum (VivaCell) at 37 °C with 5% CO2.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Competitive Binding Assay, In Vitro, Neutralization, Positive Control

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Susceptibility of various cell lines to CPE following infection by HCoVs

Article Snippet: HCT-8 cells (human ileocecal adenocarcinoma; ATCC/CCL-244), HEK 293T cells (human embryonic kidney; CRL-3216/ATCC), HEK293/ACE2-stable cell line (HEK293 cells stably expressing angiotensin-converting enzyme 2; BEI Resources/NR-52511) were grown in RPMI-1640 medium supplemented with 100 units/mL penicillin (P), 100 µg/mL streptomycin, and 10% FBS.

Techniques: Infection

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of OC43-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) OC43-infected HCT-8 cells (red). CD13, CD147, CD326, ACE2, and GD3 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected HCT-8 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of two biological replicates for all antigens except GD3, where three biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: HCT-8 cells (human ileocecal adenocarcinoma; ATCC/CCL-244), HEK 293T cells (human embryonic kidney; CRL-3216/ATCC), HEK293/ACE2-stable cell line (HEK293 cells stably expressing angiotensin-converting enzyme 2; BEI Resources/NR-52511) were grown in RPMI-1640 medium supplemented with 100 units/mL penicillin (P), 100 µg/mL streptomycin, and 10% FBS.

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of 229E-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) 229E-infected MRC-5 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected MRC-5 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD147 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: HCT-8 cells (human ileocecal adenocarcinoma; ATCC/CCL-244), HEK 293T cells (human embryonic kidney; CRL-3216/ATCC), HEK293/ACE2-stable cell line (HEK293 cells stably expressing angiotensin-converting enzyme 2; BEI Resources/NR-52511) were grown in RPMI-1640 medium supplemented with 100 units/mL penicillin (P), 100 µg/mL streptomycin, and 10% FBS.

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Phenotypic characterization of cell surface markers of NL63-infected cells. Phenotypic characterization of ( A ) uninfected (blue) and ( B ) NL63-infected LLC-MK2 cells (red). CD13, CD147, CD326, and ACE2 expression were probed by flow cytometry. ( C ) Histogram plots of control and infected LLC-MK2 cells relative to isotype control (gray). Histograms plots showing ( D ) percentage of cells expressing a given marker and ( E ) mean fluorescence intensity. Data in panels D and E represent the mean of three biological replicates, except for CD13 where two biological replicates were carried out. Statistical analysis was performed with the multiple unpaired t -test with Welch’s correction. Only results that reached statistical significance were identified: * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: HCT-8 cells (human ileocecal adenocarcinoma; ATCC/CCL-244), HEK 293T cells (human embryonic kidney; CRL-3216/ATCC), HEK293/ACE2-stable cell line (HEK293 cells stably expressing angiotensin-converting enzyme 2; BEI Resources/NR-52511) were grown in RPMI-1640 medium supplemented with 100 units/mL penicillin (P), 100 µg/mL streptomycin, and 10% FBS.

Techniques: Infection, Expressing, Flow Cytometry, Control, Marker, Fluorescence

Journal: Microbiology Spectrum

Article Title: Seasonal human coronaviruses OC43, 229E, and NL63 induce cell surface modulation of entry receptors and display host cell-specific viral replication kinetics

doi: 10.1128/spectrum.04220-23

Figure Lengend Snippet: Soluble ACE2 protein and anti-ACE2 monoclonal blocking antibody inhibit NL63 infection in LLC-MK2 cells. ( A ) Treatment with soluble ACE2 or control protein ovalbumin at 5 and 10 mg/mL was pre-incubated with NL63 virus at MOI 0.1 (virus titer: 8.89 × 10 5 TCID 50 /mL) for 1 hour, and the protein and virus mixture complex was added to the LLC-MK2 cells. ( B ) Similarly, cells were incubated for 1 hour with human anti-ACE2 monoclonal blocking antibody (10, 40, and 120 µg/mL) or unrelated control antibody (anti-DC-SIGN; 40 µg/mL) followed by addition of NL63 virus (MOI 0.1). Five days post-infection, both culture supernatants and cell lysates were harvested for quantification of nucleocapsid (N) and spike (S) vRNA copies by ddPCR. Percent relative viral inhibition following treatment with (C) sACE2 or (D) anti-ACE2 monoclonal antibody compared to their respective untreated control. Data represent three biological replicates for sACE2 or four (anti-ACE2) with three technical replicates per experiment. Statistical analysis was performed using an unpaired t -test with Welch’s correction ( P < 0.05) relative to untreated control. n.s. denoted as no statistical difference; * P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.0001, **** P ≤ 0.0001.

Article Snippet: HCT-8 cells (human ileocecal adenocarcinoma; ATCC/CCL-244), HEK 293T cells (human embryonic kidney; CRL-3216/ATCC), HEK293/ACE2-stable cell line (HEK293 cells stably expressing angiotensin-converting enzyme 2; BEI Resources/NR-52511) were grown in RPMI-1640 medium supplemented with 100 units/mL penicillin (P), 100 µg/mL streptomycin, and 10% FBS.

Techniques: Blocking Assay, Infection, Control, Incubation, Virus, Inhibition