Journal: Advanced Science

Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses

doi: 10.1002/advs.202506764

Figure Lengend Snippet: Structure analysis and computational design strategy for thermostable and proteolysis‐resistant hIFN‐λ3. a) The 3D structure of the hIFN‐λ3/hIFN‐λR1/hIL‐10Rβ (PDB code: 5T5W) [ ⁵⁰ ] complex and its downstream signaling pathway. b) Induction of representative ISG expression ( Isg15, Mx1, and Oas1 ) in HNEpCs after 12‐h treatment with recombinant hIFN‐λs WT, analyzed by RT‐qPCR ( n = 3). mRNA levels were calculated relative to non‐treated controls and normalized to human 18s rRNA expression. c) First‐derivative plots (dF/dT) of thermal shift assays for calculating melting temperatures (Tm). 12.5 µg of each hIFN‐λ was mixed with 2.5 µL of diluted Protein Thermal Shift Dye. Tm values, corresponding to the peak dF/dT temperature, are indicated. d) Surface representation of hIFN‐λ3 (PDB code: 3HHC). [ ⁴⁹ ] The structure of the α3‐4 loop, which is missing in the crystal structure, is predicted by AlphaFold2 (AF2). The binding sites of hIFN‐λ3 for IL‐10Rβ and IFN‐λR1 are highlighted in cyan and lime, respectively. The exposed hydrophobic patch (magenta), thrombin cleavage site (red), and flexible α3–4 loop (dark green) are shown. e) Targeted backbone redesign using RFdiffusion. The designated hotspot residues (59 V, 118L, and 122L) on the exposed hydrophobic patch for RFdiffusion are highlighted in light gray. The resulting 100 designed backbones are categorized by scaffold length and the number of α‐helix turns at the redesigned backbone. f) Sequence design of 45 redesigned backbones (10 sequences/1 redesigned backbone) using ProteinMPNN, followed by structure prediction with AlphaFold2 (AF2). g) Scatter plot showing AlphaFold2‐predicted confidence (pLDDT) versus RMSD (AF2 prediction compared to the generated backbone) for 450 designed hIFN‐λ3 variants. Dashed lines indicate selection thresholds (pLDDT ≥ 93, RMSD ≤ 0.8), with 221 selected designs in the lower‐right quadrant. All data represent mean ± SD from independent experiments. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test (0.01<* P <0.1, 0.001<** P <0.01, 0.0001<*** P < 0.001, **** P <0.0001 versus control; ns, not significant).

Article Snippet: For ISG induction in HNEpCs (#C‐12620, PromoCell), starved cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs (wild type, designed and heat‐incubated proteins) for 12 h. For dose‐dependent ISG induction in Vero E6 cells, cells were starved with serum‐free MEM (#LM007‐08, Welgene) for 12 h and then incubated with G‐hIFN‐λ3‐DE1 (7.8–2000 ng mL −1 ; 4‐fold serial dilution) for 24 h. Following incubation for both protocols, total RNA was extracted using Trizol reagent (#15596026, Invitrogen) following the manufacturer's protocol.

Techniques: Expressing, Recombinant, Quantitative RT-PCR, Binding Assay, Sequencing, Generated, Selection, Control

Journal: Advanced Science

Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses

doi: 10.1002/advs.202506764

Figure Lengend Snippet: Biological activity and thermal aggregation resistance of hIFN‐λ3‐DE1 under acute and long‐term heat stress. a) Relative mRNA expression of representative ISGs ( Isg15, Mx1, and Oas1 ) in HNEpCs following 12‐h treatment with hIFN‐λ3‐WT or hIFN‐λ3‐DE1 (100 ng mL −1 ), with or without short‐term heat stress (70 °C for 5 min). mRNA levels were analyzed by RT‐qPCR ( n = 3), normalized to 18s rRNA , and expressed relative to non‐treated controls. b) Short‐term thermal aggregation profiles of hIFN‐λ3‐WT and hIFN‐λ3‐DE1 after 5‐min incubation at the indicated temperatures (25, 50, 60, 70, 80, or 90 °C). Residual soluble protein concentrations were quantified (n = 3). c) Relative ISG expression ( Isg15, Mx1, and Oas1 ) in HNEpCs treated with hIFN‐λ3‐WT or hIFN‐λ3‐DE1 (100 ng mL −1 ) after long‐term incubation at 45 or 50 °C for 2 weeks. RT‐qPCR was performed as in (a) ( n = 3). d) Long‐term thermal aggregation of hIFN‐λ3‐WT and hIFN‐λ3‐DE1 during 2‐week incubation at 45 or 50 °C. Protein solubility was monitored over time ( n = 3). All data represent mean ± SD from independent experiments. Statistical analysis was performed by one‐way ANOVA followed by Sidak's multiple comparisons test (0.001<** P <0.01, 0.0001<*** P < 0.001, **** P <0.0001 vs control and ns is not significant). n.t., non‐treat; WT, hIFN‐λ3‐WT; DE1, hIFN‐λ3‐DE1.

Article Snippet: For ISG induction in HNEpCs (#C‐12620, PromoCell), starved cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs (wild type, designed and heat‐incubated proteins) for 12 h. For dose‐dependent ISG induction in Vero E6 cells, cells were starved with serum‐free MEM (#LM007‐08, Welgene) for 12 h and then incubated with G‐hIFN‐λ3‐DE1 (7.8–2000 ng mL −1 ; 4‐fold serial dilution) for 24 h. Following incubation for both protocols, total RNA was extracted using Trizol reagent (#15596026, Invitrogen) following the manufacturer's protocol.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Incubation, Solubility, Control

Journal: medRxiv

Article Title: In-vitro characterization of 2019-24 Influenza B Viruses reveals increased temperature-dependent fitness in later timepoints independent of antigenic drift

doi: 10.1101/2025.10.27.25338757

Figure Lengend Snippet: Low MOI Growth curves on human nasal epithelial cell (hNEC) cultures (MOI = 0.01) at 33°C (A) or 37°C (D) reveal significantly different kinetics in early and late time points. (B-C) Virus production at 33°C between (B) 2-48 hours post infection(hpi) or (C) 48-120 hpi. Virus production between at 37°C at (E) 2-48 hours post infection (hpi) or (F) 48-120 hpi. (G-J) Growth curves plotted by virus at 33°C and 37°C per panel. Statistical testing was performed by one way (AUC) or two-way (growth curves) ANOVA with Tukey post host test. Each graph combines two or three experiments with four hNEC wells per experiment.

Article Snippet: Human nasal epithelial cells (hNEC) (PromoCell) were cultivated as previously described.

Techniques: Virus, Infection

Journal: medRxiv

Article Title: In-vitro characterization of 2019-24 Influenza B Viruses reveals increased temperature-dependent fitness in later timepoints independent of antigenic drift

doi: 10.1101/2025.10.27.25338757

Figure Lengend Snippet: Low MOI Growth curves on human nasal epithelial cell (hNEC) cultures (MOI = 0.01) at 33°C (A) or 37°C (D) reveal significantly different kinetics in early and late time points. (B-C) Virus production at 33°C between (B) 2-48 hours post infection(hpi) or (C) 48-120 hpi. Virus production between at 37°C at (E) 2-48 hours post infection (hpi) or (F) 48-120 hpi. (G-J) Growth curves plotted by virus at 33°C and 37°C per panel. Statistical testing was performed by one way (AUC) or two-way (growth curves) ANOVA with Tukey post host test. Each graph combines two or three experiments with four hNEC wells per experiment.

Article Snippet: Human nasal epithelial cells (hNEC) (PromoCell) were cultivated as previously described.

Techniques: Virus, Infection