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: bioRxiv

Article Title: Staged heavy-chain filtering enables Fab discovery from combinatorially intractable library spaces

doi: 10.64898/2026.05.10.724059

Figure Lengend Snippet: (a, b) Binding ELISA curves and apparent half-maximal effective concentration (EC₅₀) values of CLR101 and reference antibodies (CR3022, P2B-2F6, and S309) against the SARS-CoV-2 D614G spike protein (a) and wild-type RBD (b) (n = 2 independent experiments, mean ± s.d.). (c) Apparent EC₅₀ values of CLR101, CR3022, P2B-2F6, and S309 against RBDs of six SARS-CoV-2 variants—wild-type (Wuhan-Hu-1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and BA.5 (B.1.1.529.5)—determined by ELISA (n = 2 independent experiments, mean ± s.d.). (d) Epitope binning analysis of CLR101 by competitive ELISA. The heatmap displays the mean percent inhibition of CLR101 binding to wild-type RBD in the presence of excess competitor proteins (ACE2, CLR101, CR3022, P2B-2F6, and S309). Self-competition by CLR101 was included as a positive control for binding inhibition. The color scale indicates the degree of inhibition from 0% to 100% (n = 3 independent experiments). (e, f) Evaluation of in vitro neutralizing activity against D614G spike-pseudotyped lentiviral particles using hACE2-293T cells. (e) Dose-response neutralization curve of CLR101, showing neutralizing activity with an apparent EC₅₀ of 11.1 ± 2.6 nM (n = 2 independent experiments, mean ± s.d.). (f) Percent neutralizing activity of CLR101 alongside benchmark antibodies at a fixed antibody concentration of 100 nM (n = 2 independent experiments, mean ± s.d.).

Article Snippet: Virus–antibody mixtures were then added to monolayers of hACE2-expressing 293T cells (hACE2-293T; Takara Bio Inc., Kusatsu, Shiga, Japan) in 96-well plates.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Competitive ELISA, Inhibition, Positive Control, In Vitro, Activity Assay, Neutralization

Journal: iScience

Article Title: CGM23 corresponds to a pan-coronavirus lipopeptide inhibitor potently inhibiting virion fusion

doi: 10.1016/j.isci.2026.115334

Figure Lengend Snippet: Peptide design and evaluation (A) Schematic representation of SARS-CoV-2 spike protein and amino acid sequences of HR2 peptides from SARS-CoV-2 and EK1 (HCoV-OC43 HR2 derived peptide). N , N-terminus ; C , C-terminus ; S1/S2 , cleavage site at S1/S2 boundary ; RBD , receptor-binding domain ; HR1 , heptad repeat 1 ; HR2 , heptad repeat 2 ; HR2P , heptad repeat 2 peptide. Syncytia assay. Left: GFP- and spike-expressing 293T cells were co-cultured with RFP- and ACE2-expressing Calu-6 cells for 16 h in the presence or absence of peptides. Center: double-positive cells (white arrows) indicative of syncytia formation are frequent in the presence of the scrambled EK1 peptide (Ⅰ) but not in the presence of CGM23 (Ⅱ) (100 nM). Right: quantification of syncytia formation in the presence of the scrambled EK1 peptide (top) or CGM23 (bottom) relative to mock treatment. (B) Sequence, N- and C-terminal modifications and IC 50 of the 15 peptides with IC 50 s < 10 nM in the syncytia assay. CoV-2, SARS-CoV-2; N-term, N-terminus; C-term, C-terminus. IC 50 data are means of samples from a representative experiment. Ac, acetylation; PPA, 4-phenylpropanoic acid; PBA, 4-phenylbutanonic acid. (C) Correlation between IC 50 values in the pseudotyped SARS-CoV-2 spike virion assay and live SARS-CoV2 virus infection assay for the 15 peptides with IC 50 values below 10 nM in the syncytia assay. Statistical analysis was performed using Spearman’s rank test. (D and E) Dose-dependent inhibitory activity of CGM23 and EK1C4 relative to CG167 (EK1 scrambled peptide with EK1C4 lipidation) in the pseudotyped SARS-CoV2 spike virion assay (D) and live SARS-CoV2 infection assay (E). IC 50, half-maximal inhibitory concentration.

Article Snippet: Live virus experiments were performed with Calu-6 epithelial cells (ATCC HTB-56) stably expressing human Angiotensin Converting Enzyme 2 (ACE2) (OriGene, RC08442) as target cells.

Techniques: Derivative Assay, Binding Assay, Expressing, Cell Culture, Sequencing, Virus, Infection, Activity Assay, Concentration Assay

Journal: iScience

Article Title: CGM23 corresponds to a pan-coronavirus lipopeptide inhibitor potently inhibiting virion fusion

doi: 10.1016/j.isci.2026.115334

Figure Lengend Snippet: In vivo prophylactic and therapeutic efficacy of CGM23 against SARS-CoV-2 live virus in mice (A) The SARS-CoV-2 outgrowth assay. Lung homogenates were collected 2 days post viral infection (DPI) combined with intranasal administration of CGM23 and EK1C4 (12.5 μg, 0.865 mg/kg). (B) Diluted lung homogenates were added to Calu-6-ACE2 cells and infection titers were measured by the luciferase assay 48 h later. CG167, EK1 scrambled peptide with EK1C4 lipidation. Data presented correspond to mean ± SD. ∗ p < 0.05. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (C) Histopathological findings of mouse lungs at 2 days after virus inoculation. Images are shown clockwise starting from the top left: CG167 group, EK1C4 group, non-infected group, and CGM23 group. Lung sections were stained with anti-spike antibody (green) and anti-MAC-2 antibody (magenta) and DAPI (blue). Scale bars, 100μm. (D and E) Quantitative analysis of lung histopathological findings for each group. Data shown represent means ± SD. (F) Therapeutic treatment. CGM23 and EK1C4 were administered intranasally (12.5 μg, 0.865 mg/kg; 25 μg, 1.73 mg/kg) 8 h after SARS-CoV-2 inoculation, and lung homogenates were collected 24 h later for plaque assay analysis. Data presented correspond to mean ± SD. ∗ p < 0.05, ∗∗∗ p < 0.001. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (G) Prophylactic treatment. SARS-CoV-2 was administered intranasally 30 min after intranasal administration of CGM23 or EK1C4 (12.5 μg, 0.865 mg/kg; 25 μg, 1.73 mg/kg). Lung tissues were collected 24 h later for plaque assay analysis.

Article Snippet: Live virus experiments were performed with Calu-6 epithelial cells (ATCC HTB-56) stably expressing human Angiotensin Converting Enzyme 2 (ACE2) (OriGene, RC08442) as target cells.

Techniques: In Vivo, Drug discovery, Virus, Infection, Luciferase, Staining, Plaque Assay