Journal: International Journal of Molecular Sciences

Article Title: Immunogenicity and Protection of mRNA Vaccine Encoding Spike Protein of SARS-CoV-2 Omicron-XEC Subvariant

doi: 10.3390/ijms27104218

Figure Lengend Snippet: Construction and characterization of the XEC-S mRNA vaccine. ( A ) The XEC-S mRNA was constructed to encode the ectodomain S (S1 and S2 subunits) protein of the Omicron-XEC subvariant of SARS-CoV-2 with the HexaPro sequence, and to contain an N-terminal tissue plasminogen activator (tPA signal peptide), a C-terminal foldon trimeric sequence and a His 6 tag. The synthesized mRNA carrying a 5′-untranslated region (5′-UTR) and a 3′-UTR was capped at the 5′-terminus and tailed with a poly(A) sequence at the 3′-terminus, then encapsulated with lipid nanoparticles (LNPs) to form XEC-S-mRNA LNPs. Measured stability of the LNP-formulated XEC-S-mRNA ( B ) and control LNPs ( C ) by a DynaPro NanoStar II Light Scattering Detector (DLS) instrument. The samples were stored at 4 °C, 25 °C, and 37 °C for 1 to 7 days, then the particle sizes (diameters) were measured by the DLS. Histograms showing particle sizes of the LNP-formulated XEC-S-mRNA ( D ) and control LNPs ( E ). ( F ) Assessment of the expression of the His-tagged protein encoded by XEC-S-mRNA using flow cytometry. HEK293T cells were incubated with XEC-S mRNA-LNPs or the control LNPs, then stained with the anti-His-FITC antibody prior to conducting fluorescence intensity analysis using a flow cytometer. The shaded region indicates control cells incubated with LNPs, and the magenta line refers to target cells incubated with LNP-formulated XEC-S-mRNA. MFI: median fluorescence intensity.

Article Snippet: HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO 2 .

Techniques: Construct, Sequencing, Synthesized, Control, Expressing, Flow Cytometry, Incubation, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Immunogenicity and Protection of mRNA Vaccine Encoding Spike Protein of SARS-CoV-2 Omicron-XEC Subvariant

doi: 10.3390/ijms27104218

Figure Lengend Snippet: Assessment of humoral immune responses induced by the XEC-S-mRNA vaccine. ( A ) Immunization and challenge schedules. BALB/c-hACE2 transgenic mice were intradermally (i.d.) immunized with LNP-formulated XEC-S-mRNA or control LNPs and boosted twice at 3-week intervals; collection of sera followed 10 days after the last dose for measurement of subsequent antibody responses. Nine weeks after the last dose, the immunized mice were then intranasally (i.n.) challenged with an Omicron-KP.3 subvariant of SARS-CoV-2 to assess the protective efficacy. Evaluation of the XEC-S-specific IgG ( B ), IgG1 ( C ), and IgG2a ( D ) antibody (Ab) titers in sera by ELISA. The data refers to the mean ± standard deviation of the mean (s.e.m) of five mice in each group. The dotted lines indicate the detection limit (1:30). The experiments were repeated once, with similar results obtained.

Article Snippet: HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO 2 .

Techniques: Transgenic Assay, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Immunogenicity and Protection of mRNA Vaccine Encoding Spike Protein of SARS-CoV-2 Omicron-XEC Subvariant

doi: 10.3390/ijms27104218

Figure Lengend Snippet: Evaluation of the broad neutralizing antibody responses induced by the XEC-S-mRNA vaccine. Mouse sera collected 10 days after the third immunization were assessed for a neutralizing antibody (Ab) titer against pseudotyped Omicron-KP.2 ( A ), KP.3 ( B ), XEC ( C ), NB.1.8.1 ( D ), and XFG ( E ) using a pseudovirus neutralization assay. The same sera were assessed for a neutralizing Ab titer against the infection of live SARS-CoV-2 Omicron subvariants, including KP.2 ( F ) and KP.3 ( G ), using a cytopathic effect (CPE)-based neutralization assay. The NT 50 (i.e., 50% neutralizing Ab titer) is shown as the mean ± s.e.m of five mice in each group. The dotted lines indicate the detection limit (1:60 for the pseudovirus neutralizing Ab titer, and 1:30 for the live virus neutralizing Ab titer). The experiments were repeated once, with similar results obtained.

Article Snippet: HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO 2 .

Techniques: Neutralization, Infection, Virus

Journal: International Journal of Molecular Sciences

Article Title: Immunogenicity and Protection of mRNA Vaccine Encoding Spike Protein of SARS-CoV-2 Omicron-XEC Subvariant

doi: 10.3390/ijms27104218

Figure Lengend Snippet: The LNP-formulated XEC-S-mRNA vaccine protected against a SARS-CoV-2 Omicron-KP.3 challenge. Nine weeks after the final immunization, the BALB/c-hACE2 transgenic mice were challenged (i.n.) with an Omicron-KP.3 subvariant of SARS-CoV-2, then viral titers in the lungs ( A ) and trachea ( B ) were measured by means of the plaque assay 5 days post-challenge. The data (plaque-forming unit: PFU/mL of viral titers) is shown as the mean ± s.e.m of five mice in each group. The dotted lines indicate the detection limit (3.3 PFU/mL). The unpaired Student’s t test was used to analyze statistical significance between the XEC-S-mRNA and control LNP groups. ** indicates p < 0.01. The experiments were repeated once, with similar results obtained.

Article Snippet: HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO 2 .

Techniques: Transgenic Assay, Plaque Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Immunogenicity and Protection of mRNA Vaccine Encoding Spike Protein of SARS-CoV-2 Omicron-XEC Subvariant

doi: 10.3390/ijms27104218

Figure Lengend Snippet: XEC-S-mRNA-induced neutralizing antibodies play a key role in the protection against a SARS-CoV-2 Omicron-KP.3 challenge. ( A ) Immunization and serum transfer schedules. BALB/c mice were immunized (i.d.) with LNP-formulated XEC-S-mRNA or control LNPs and boosted at 3, 6, and 22 weeks. The pooled sera collected at 10, 17, and 28 days after the last dose were assessed for neutralizing antibody (Ab) titers against pseudotyped ( B ) and live ( C ) Omicron-KP.3 subvariants of SARS-CoV-2, then injected (i.p.) into naïve B6-hACE2 transgenic mice. 6 h post serum-transfer, the mice were challenged (i.n.) with Omicron-KP.3; five days post-challenge, the lungs ( D ) and trachea ( E ) were collected and assessed for viral titers using the plaque assay. The NT 50 indicates a 50% neutralizing Ab titer, and the viral titer is expressed as PFU/mL. The data is shown as the mean ± s.e.m of duplicate or quadruple wells (for the pooled sera) or of five mice in each group (for the viral titer). The dotted lines indicate the detection limit (1:60 for the pseudovirus neutralizing Ab titer, 1:30 for the live virus neutralizing Ab titer, and 3.3 PFU/mL for the viral titer). The unpaired Student’s t test was used to analyze the statistical significance between the XEC-S-mRNA and control LNP groups. * and **** indicate p < 0.05 and p < 0.0001, respectively. The experiments were repeated once, with similar results obtained.

Article Snippet: HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO 2 .

Techniques: Control, Injection, Transgenic Assay, Plaque Assay, Virus

Journal: ImmunoHorizons

Article Title: IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting ACE2-mediated virus binding and cell entry

doi: 10.1093/immhor/vlaf056

Figure Lengend Snippet: IL-13 inhibits ACE2 transcription in epithelial cells. ACE2 mRNA levels were measured by RT-qPCR in Vero E6 epithelial cells treated with IL-13 (10 ng/ml) or PBS for 24, 48, or 72 h (A), or in Calu3 cells treated with IL-13 (10 ng/ml) or PBS for 24 h (B), or in human primary nasal (C) and bronchial (D) epithelial cells treated with IL-13 (10 ng/ml) or PBS for 24 h. The mRNA levels at each time point were normalized by GAPDH (A, B) or PPIA (C, D) and are shown relative to PBS. Shown are means ± SEM from 1 representative experiment out of 2. Each data point represents a replicate culture [ n = 5–6/condition (A), n = 3/condition (B)]. In (C, D) each data point represents a single donor ( n = 8–12) each tested in triplicate. An unpaired 2-tailed t test was used for statistical analysis. An unpaired 2-tailed t test was used for statistical analysis.

Article Snippet: HEK293T/ACE2 cells stably transduced with human ACE2 were purchased from GeneCopoeia (SL221, Rockville, Maryland, USA).

Techniques: Quantitative RT-PCR

Journal: ImmunoHorizons

Article Title: IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting ACE2-mediated virus binding and cell entry

doi: 10.1093/immhor/vlaf056

Figure Lengend Snippet: IL-13 downregulates surface ACE2 expression in epithelial cells. Vero (A, B) or Calu-3 (C) cells were treated with IL-13 (2 or 10 ng/ml) for 72 h and assessed for ACE2 protein expression by flow cytometry using ACE2-specific monoclonal antibodies (clone 36 from Sino Biological in A and B, clone 535919 from R&D systems in C) and relevant isotype controls. Shown are a representative flow cytometry histogram for Vero cells (A) (shaded area: isotype control) and proportions of ACE2 + cells in IL-13-treated cells relative to PBS (B, C). The data are from 1 representative experiment out of 3 and show means ± SEM. Data points represent replicate cultures ( n = 4/condition). An unpaired 2-tailed t test was used for statistical analysis.

Article Snippet: HEK293T/ACE2 cells stably transduced with human ACE2 were purchased from GeneCopoeia (SL221, Rockville, Maryland, USA).

Techniques: Expressing, Flow Cytometry, Bioprocessing, Control

Journal: ImmunoHorizons

Article Title: IL-13 protects epithelial cells from SARS-CoV-2 infection by inhibiting ACE2-mediated virus binding and cell entry

doi: 10.1093/immhor/vlaf056

Figure Lengend Snippet: IL-13 does not affect ACE2 expression and S protein-pseudotyped lentiviral entry into HEK293T/ACE2 cell transfectants. HEK293T/ACE2 cells were treated with 10 ng/ml IL-13 for 72 h and surface ACE2 expression was evaluated by flow cytometry with ACE2-specific monoclonal antibodies. Shown are representative flow cytometry histograms (A), proportions (B) and MFI (C) of ACE2+ cells in cultures treated with IL-13 or PBS. Data are from 1 experiment ( n = 5–7 replicates per condition). An unpaired, two-tailed t test was used for statistical analysis. ACE2 -transfected HEK293T/ACE2 cells were treated with IL-13 (10 ng/ml) (E, H) or PBS (D, G) for 48 h, washed and transduced with lentiviral particles pseudotyped with SARS-CoV-2 S protein (0.2 × 10 4 TU: D, E) or VSV-G protein (2 × 10 4 TU: G, H). Shown are frames representative of GFP fluorescence under the indicated experimental conditions. GFP fluorescence was assessed under a microscope (10× magnification) 72 h post transduction and quantified using Slide Analyzer (F, I). The scale bars represent 200 µm. Results represent the mean ± SEM of 20 (SARS-CoV-2-S-pseudotyped virus) or 10 (VSV-G-pseudotyped virus) random frames from 1 out of 2 representative experiments. A Wilcoxon 2-sample test was used for statistical analysis.

Article Snippet: HEK293T/ACE2 cells stably transduced with human ACE2 were purchased from GeneCopoeia (SL221, Rockville, Maryland, USA).

Techniques: Expressing, Flow Cytometry, Bioprocessing, Two Tailed Test, Transfection, Transduction, Fluorescence, Microscopy, Virus