vero cells  (ATCC)

Journal: Molecular Therapy Oncology

Article Title: A measles virus encoding a CD19/CD3 bispecific T cell engager shows enhanced preclinical anti-BCP-ALL efficacy without significant toxicity

doi: 10.1016/j.omton.2026.201127

Figure Lengend Snippet: MV-Blina, an oncolytic measles virus encoding the CD19/CD3-bispecific T cell engager, replicates and is cytotoxic (A) Schematic of MV-Blina. Genomes of the parental MV-Edm strain and the recombinant MV-Blina. The bsTE construct contains the anti-human-CD19 variable heavy/light domain (CD19 V H/L ) and the anti-human-CD3 variable heavy/light domain (CD3 V H/L ). (B) Enhanced replication of MV-Blina compared to MV-Edm. Replication of MV-Blina was assessed by infecting Vero cells at MOI 0.01 or 1.0 with MV-Blina and MV-Edm, respectively. Viral titers at the indicated time points were determined with a titration assay. Results are shown as means ± SD of n = 3 from independent experiments seeded in duplicates. (C) Comparable cytotoxicity of MV-Blina and parental MV-Edm. Vero cells were infected with MOI 0.01, 0.1, or 1.0 of MV-Blina and MV-Edm, respectively. Cell viability was determined using the MTT assay at the indicated time points. Results are shown as means ± SD of n ≥ 3 from independent experiments seeded in sextuplicates. Statistical analysis was performed using two-way ANOVA with Dunnett’s correction. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.

Article Snippet: The recombinant MV-bsTE virus was generated by transfecting Vero cells with antigenomic cDNA constructs as described previously., The MV-Edmonston B strain (VR-24, ATCC, Manassas, VA, USA) was obtained and amplified prior to experiments.

Techniques: Virus, Recombinant, Construct, Titration, Infection, MTT Assay

Journal: Molecular Therapy Oncology

Article Title: A measles virus encoding a CD19/CD3 bispecific T cell engager shows enhanced preclinical anti-BCP-ALL efficacy without significant toxicity

doi: 10.1016/j.omton.2026.201127

Figure Lengend Snippet: MV-Blina infected cells secrete target-binding secBlina, which initiates ALL cell kill more efficiently than CD19/CD3-bsTE (A) Vero cells secrete secBlina. secBlina was harvested from the supernatant of infected Vero cells, followed by purification and concentration. Eluate (2 μg protein) was detected via His-tag by Western Blot in comparison to the commercial CD19/CD3-bsTE (1.8 μg). (B) Purified and concentrated secBlina binds specifically to target cells. Binding to target cells was confirmed by flow cytometry using CD3 − CD19 + REH, CD3 + CD19 - Jurkat, and CD3 − CD19 - K562 cell lines incubated with secBlina. Binding of secBlina to target cells was detected by flow cytometry via a His-tag antibody. (C) secBlina strongly binds to REH target cells. The REH cell line was first incubated with 1 μg secBlina. Increasing concentrations of CD19/CD3-bsTE were then added to displace cell-bound secBlina. Cell-bound secBlina was detected via an HA tag antibody by flow cytometry. (D) T cells in human PBMCs are activated by secBlina. Pooled PBMCs were stimulated with an (E) T cells are activated by secBlina during co-culture of PBMCs with REH cells. Pooled PBMCs were co-cultured with REH cells in a 1:1-ratio for 24 h while being treated with secBlina or the CD19/CD3-bsTE. Activated CD2 + CD69 + T cells were detected by flow cytometry. (F) MV-Blina infected leukemia cells produce and secrete secBlina. REH and Jurkat cells are shown 72 h post MV infection or Opti-MEM control. secBlina was detected by His-tag IF staining. Scale bars 50 μm. (G) secBlina effectively induces REH target cell kill compared to CD19/CD3-bsTE, whereas K562 control cells are unaffected. Target (T) cells REH (left panels) and negative control cells K562 (right panels) were incubated with secBlina (green) or CD19/CD3-bsTE (blue) for 24 h at indicated concentrations in the presence of PBMCs (effector; E) at different E:T ratios. Specific cell kill was determined by flow cytometry. Results in A, B, C, and F are shown as representatives of three independent experiments. Data are shown as mean ± SD of n = 3 (D, E) or n = 5 (G) independent experiments. Statistical analysis was performed using two-way ANOVA with Dunnett’s correction. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; and ∗∗∗∗, p < 0.0001.

Article Snippet: The recombinant MV-bsTE virus was generated by transfecting Vero cells with antigenomic cDNA constructs as described previously., The MV-Edmonston B strain (VR-24, ATCC, Manassas, VA, USA) was obtained and amplified prior to experiments.

Techniques: Infection, Binding Assay, Purification, Concentration Assay, Western Blot, Comparison, Flow Cytometry, Incubation, Co-Culture Assay, Cell Culture, Control, Staining, Negative Control

Journal: iScience

Article Title: FGFR signaling and neddylation facilitate SARS-CoV-2 infection by modulating interferon induction and viral entry, respectively

doi: 10.1016/j.isci.2025.114566

Figure Lengend Snippet: Inhibiting FGFR signaling enhances the interferon response (A) HEK293T-ACE2 cells were transfected with a SARS-CoV-2 nanoluciferase reporter replicon. Then, the cells were treated with MLN4924 (100 nM), BGJ398 (10 μM), remdesivir (10 μM), IFNα (100 unit/mL), or DMSO alone. Luminescence (expressed as arbitrary units) was measured at 24 and 48 h post-treatment. (B) Schematic of FGFR and downstream signaling pathways and inhibitors used to block them (indicated in red). (C) HEK293T-ACE2 cells were pretreated with the indicated inhibitors of signaling pathways downstream of FGFR for 24 h and then infected with SARS-CoV-2 (72B/CA/CALG) using an MOI of 0.1. Twenty-four hours later, media was collected and subjected to the plaque assay to determine viral titers. (D) Normal human bronchial epithelial (NHBE) cells were treated with DMSO alone, MLN4924, or BGJ398 at 1 μM for 24 h and then infected with 400 HAU/mL of Sendai virus. Total cellular RNA was harvested 4 or 8 h post infection (hpi) and then subjected to qRT-PCR to determine relative levels of mRNA encoding type I IFNβ. (E) NHBE cells were treated as described in (D). Total cellular RNA was harvested 8 hpi and then subjected to qRT-PCR to determine relative levels of mRNA encoding ISGs (OAS1, MX1, and MX2). (F) Vero cells were pretreated with MLN4924 (10 μM), BGJ398 (100 μM), remdesivir (10 μM), or DMSO for 24 h and then infected with SARS-CoV-2 (72B/CA/CALG), using an MOI of 0.01. Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. Data shown are averaged from three independent experiments. Error bars represent standard error of the mean. One-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance between the control (DMSO) and drug-treated samples. p value < 0.01 ∗∗, <0.001 ∗∗∗, <0.0001 ∗∗∗∗, ns = not significant (A, C, D, E, and F).

Article Snippet: Vero CCL81, HEK 293T, HEK293FT, Vero E6, Huh 7.5, A549 and Calu-3 cells were sourced from the American Type Culture Collection (Manassas, VA), Vero-TMPRSS2 from JCRB cell bank 1819, and HEK 293T-ACE2 and NCI-H23-ACE2 were developed in-house by stable transduction of ACE2 expressing lentivirus constructs.

Techniques: Transfection, Protein-Protein interactions, Blocking Assay, Infection, Plaque Assay, Virus, Quantitative RT-PCR, Comparison, Control

Journal: iScience

Article Title: FGFR signaling and neddylation facilitate SARS-CoV-2 infection by modulating interferon induction and viral entry, respectively

doi: 10.1016/j.isci.2025.114566

Figure Lengend Snippet: An internal deletion within the spike protein confers resistance to the neddylation inhibitor MLN4924 (A) SARS-CoV-2 (72B/CA/CALG) was passaged in Vero cells in the presence of MLN4924 or DMSO alone. For passage 1, an MOI of 0.01 was used for infection. Twenty microliters of media from infected cells were then used to infect a new batch of cells after 72 h or when 40% of the cells showed CPE. (B) Vero cells were pretreated with DMSO or MLN4924 (10 μM) for 24 h and then infected with aliquots of serially passaged SARS-CoV-2 (72B/CA/CALG) (MOI of 0.01) as explained in (A) for each passage. Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. (C) HEK293T-ACE2 cells were pretreated with DMSO or MLN4924 or TAS4464 both at 100 nM for 24 h and then infected with SARS-CoV-2-WT or a SpikeΔ9 strain (MOI = 0.1). Twenty-four hours later, the media was collected and subjected to the plaque assay to determine viral titers. Data shown are averaged from three independent experiments. Error bars represent standard error of the mean. One-way ANOVA with Dunnett’s multiple comparison test was used to determine statistical significance between the control (DMSO) and drug-treated samples. p value < 0.05 ∗, <0.01 ∗∗, <0.0001 ∗∗∗∗, ns = not significant (B and C). (D) Titers from plaque assays conducted on media from Vero cells electroporated with infectious DNA clones for WT and SpikeΔ9. Error bars represent standard error of the mean. (E) Structural analysis of the spike protein deletion mutant NTD Δ68–76 associated with MLN4924 resistance. Left: mutant spike trimer model with one monomer highlighted (NTD in slate color, RBD in green). Right: NTD domains of wild type (slate) and the deletion mutant (wheat). In the wild-type spike, AlphaFold2 predicts that residues 68–76 (cyan sticks) form a disordered loop-like structure that is absent in cryo-EM structures. In NTDΔ68–76, the missing loop is indicated by an arrow. Residues A67 and K68 (corresponding to K77 in the NTD-WT) are shown in green. Despite the deletion, the overall NTD structure is preserved in the mutant. (F) Analysis of the spike NTDΔ68–76 bound to N12-9 Fab antibody (PDB: 7V23 ). The left image shows the spike trimer with one monomer (deep salmon) bound to the antibody. The antibody’s heavy and light chains bound to the NTD are colored in limon and deep olive, respectively. The right inset overlays the NTDΔ68–76 modeled using AlphaFold2 (wheat), with the NTDΔ68–76 bound to the antibody (PDB: 7V23 , deep salmon). Antibody binding to the NTD induces movement of the Tyr248–Ser256 loop, as indicated by the dotted arrow. In the absence of the disordered loop (Δ68–76), the bordering residues His66, Ala67, Lys68, and Arg69 connect to form a more ordered structure as shown in green. This conformation is consistent between the cryo-EM and AlphaFold2 models. The green ball-and-stick representation in the bottom image highlights the network of interactions formed by these residues. Atoms involved in these interactions are labeled in black, with hydrogen bonds (black) and stacking interactions (pink) shown as dotted lines.

Article Snippet: Vero CCL81, HEK 293T, HEK293FT, Vero E6, Huh 7.5, A549 and Calu-3 cells were sourced from the American Type Culture Collection (Manassas, VA), Vero-TMPRSS2 from JCRB cell bank 1819, and HEK 293T-ACE2 and NCI-H23-ACE2 were developed in-house by stable transduction of ACE2 expressing lentivirus constructs.

Techniques: Infection, Plaque Assay, Comparison, Control, Clone Assay, Mutagenesis, Cryo-EM Sample Prep, Binding Assay, Labeling