Journal: NPJ Vaccines

Article Title: A live attenuated NS1-deficient vaccine candidate for cattle-origin influenza A (H5N1) clade 2.3.4.4.b viruses

doi: 10.1038/s41541-025-01207-9

Figure Lengend Snippet: a Schematic representation of the IFNβ induction bioassay: MDCK cells constitutively expressing GFP-CAT and FFluc reporter genes under the control of the IFNβ promoter (MDCK IFNβ GFP-CAT/FFluc) were infected at MOI 2 with LPhTX, LPhTXdNS1, or mock-infected. At 12 hpi, IFNβ promoter activation was determined by GFP ( b ) and FFluc ( c ) expression. The viral NP in infected cells was detected with the HT103 MAb and DAPI was used for nuclear staining. The scale bar was set for 100 μm. BF: Bright-Field; GFP: Green Fluorescent Protein. All data points are shown with black lines representing the mean ( n = 3). Groups compared using a Welch’s one-way ANOVA, followed by multiple comparisons using Dunnett T3 method (*** = p < 0.001; **** = p < 0.0001).

Article Snippet: For immunostaining, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at RT and stained with the mouse monoclonal antibody (MAb) HT103 against influenza viral nucleoprotein (NP) and the Vectastain ABC kit (Vector Laboratories, USA), following the manufacturer’s protocol.

Techniques: Bioassay, Expressing, Control, Infection, Activation Assay, Staining

Journal: mBio

Article Title: A decavalent composite mRNA vaccine against both influenza and COVID-19

doi: 10.1128/mbio.00668-24

Figure Lengend Snippet: Design and characterization of 10-valent mRNA vaccine (FLUCOV-10). ( A ). Schematic illustration of the FLUCOV-10 formulation, a 10-valent combination mRNA vaccine targeting both influenza and COVID-19. It includes mRNAs encoding the full-length HA proteins from influenza A virus subtypes A/H1, A/H3, A/H5, and A/H7, and from influenza B virus lineages B/Yamagata and B/Victoria. Additionally, it encodes full-length spike proteins from SARS-CoV-2 variants Wuhan-Hu-1, BQ.1.1, BA.2.75.2, and XBB.1.5. Each mRNA component is individually encapsulated in lipid nanoparticles (LNPs) prior to being combined into the final FLUCOV-10 formulation. (B and C) Phylogenic trees were created for influenza HAs ( B ) and SARS-CoV-2 spikes ( C ) by using Nextstrain. The vaccine HAs or spike are indicated with red triangles, and the challenge viruses are indicated with an “X.” (D) The expression of FLUCOV-10-mRNA-encoded HA proteins in 293T cells was determined by western blotting. Lane 1, 293T cells with mock transfection; lane 2, 293T cells with indicated mRNA transfection. (E) The expression of FLUCOV-10-mRNA-encoded spike proteins. Lane 1, 293T cells with mock transfection; lane 2–5, 293T cells with Wuhan-Hu-1, BQ.1.1, BA.2.75.2, and XBB.1.5 mRNA transfection, respectively. β-Actin was used as western blotting loading control. (F) Particle size distribution of individual LNP formulations was measured in triplicate using dynamic light scattering (DLS) on the NS-90Z Nanoparticle Size and Zeta Potential Analyzer.

Article Snippet: The HA and spike proteins in cell lysates were then detected by western blotting using a mouse monoclonal antibody against SARS-CoV-2 spike proteins (GTX632604, GeneTex), a rabbit polyclonal antibody against influenza A/H1 HA (11055-T62, Sino Biological), a mouse monoclonal antibody against influenza A/H3 HA (11056-MM03, Sino Biological), a rabbit polyclonal antibody against influenza A/H5 HA (11062-T62, Sino Biological), a rabbit polyclonal antibody against influenza A/H7 HA (40103-T62, Sino Biological), a rabbit polyclonal antibody against influenza B/Yamgata lineage HA (11053-T62, Sino Biological), and a mouse monoclonal antibody against Influenza B/Victoria lineage HA (11053-MM06, Sino Biological). β-Actin was detected using anti-β-actin antibody.

Techniques: Formulation, Virus, Expressing, Western Blot, Transfection, Control, Zeta Potential Analyzer

Journal: Poultry Science

Article Title: Chicken intestinal organoids reveal polarity-dependent replication dynamics and immune responses of low pathogenic avian influenza viruses

doi: 10.1016/j.psj.2025.104921

Figure Lengend Snippet: Viral Replication Kinetics in Ba-o and Ap-o CIOs. The viral replication kinetics of the H9N2-R66 and H6N1-2904 strains in Ba-o, Ap-o CIOs, and Caco2 organoids. All CIOs were inoculated directly with LPAIVs (H9N2-R66 and H6N1-2904) without the addition of TPCK-treated trypsin (w/o TPCK). In contrast, Caco-2 organoids, including both Ap-o and Ba-o forms, were infected either with (w TPCK) or without (w/o TPCK) the addition of TPCK-treated trypsin during the experiments. Ba-o CIOs supported higher viral titers (up to 10 5 FFU/ml) compared to Ap-o CIOs, emphasizing the influence of the epithelial orientation on viral replication dynamics (A-B). The results were shown as means ± SD of nine CIOs (Ap-o & Ba-o). Statistical comparisons of viral titers between H9N2-R66 and H6N1-2904 in Ap-o and Ba-o organoids at different time points were performed using Tukey's multiple comparisons test (*** P < 0.001, ** P < 0.01, * P < 0.05). Immunofluorescence staining of virus nucleoprotein in CIOs on day 1 post-H6N1-2904 infection was visualized (Ap-o CIO: C, Ba-o CIO: D). The monoclonal mouse antibodies against influenza viral nucleoprotein were visualized by recognizing an Alexaflour 488 conjugated secondary antibody (NP, Green), and villi (red) were visualized to represent the location of the viral particles. Scale bar: 100µm.

Article Snippet: Following permeabilization, the cells were incubated with a mouse monoclonal antibody against influenza nucleoprotein (Bio-Rad) and subsequently treated with an HRP-conjugated goat anti-mouse IgG secondary antibody (SeraCare KPL).

Techniques: Infection, Immunofluorescence, Staining, Virus

Journal: mBio

Article Title: A decavalent composite mRNA vaccine against both influenza and COVID-19

doi: 10.1128/mbio.00668-24

Figure Lengend Snippet: Design and characterization of 10-valent mRNA vaccine (FLUCOV-10). ( A ). Schematic illustration of the FLUCOV-10 formulation, a 10-valent combination mRNA vaccine targeting both influenza and COVID-19. It includes mRNAs encoding the full-length HA proteins from influenza A virus subtypes A/H1, A/H3, A/H5, and A/H7, and from influenza B virus lineages B/Yamagata and B/Victoria. Additionally, it encodes full-length spike proteins from SARS-CoV-2 variants Wuhan-Hu-1, BQ.1.1, BA.2.75.2, and XBB.1.5. Each mRNA component is individually encapsulated in lipid nanoparticles (LNPs) prior to being combined into the final FLUCOV-10 formulation. (B and C) Phylogenic trees were created for influenza HAs ( B ) and SARS-CoV-2 spikes ( C ) by using Nextstrain. The vaccine HAs or spike are indicated with red triangles, and the challenge viruses are indicated with an “X.” (D) The expression of FLUCOV-10-mRNA-encoded HA proteins in 293T cells was determined by western blotting. Lane 1, 293T cells with mock transfection; lane 2, 293T cells with indicated mRNA transfection. (E) The expression of FLUCOV-10-mRNA-encoded spike proteins. Lane 1, 293T cells with mock transfection; lane 2–5, 293T cells with Wuhan-Hu-1, BQ.1.1, BA.2.75.2, and XBB.1.5 mRNA transfection, respectively. β-Actin was used as western blotting loading control. (F) Particle size distribution of individual LNP formulations was measured in triplicate using dynamic light scattering (DLS) on the NS-90Z Nanoparticle Size and Zeta Potential Analyzer.

Article Snippet: The HA and spike proteins in cell lysates were then detected by western blotting using a mouse monoclonal antibody against SARS-CoV-2 spike proteins (GTX632604, GeneTex), a rabbit polyclonal antibody against influenza A/H1 HA (11055-T62, Sino Biological), a mouse monoclonal antibody against influenza A/H3 HA (11056-MM03, Sino Biological), a rabbit polyclonal antibody against influenza A/H5 HA (11062-T62, Sino Biological), a rabbit polyclonal antibody against influenza A/H7 HA (40103-T62, Sino Biological), a rabbit polyclonal antibody against influenza B/Yamgata lineage HA (11053-T62, Sino Biological), and a mouse monoclonal antibody against Influenza B/Victoria lineage HA (11053-MM06, Sino Biological). β-Actin was detected using anti-β-actin antibody.

Techniques: Formulation, Virus, Expressing, Western Blot, Transfection, Control, Zeta Potential Analyzer