Journal: Nature Communications

Article Title: RNA replicon vaccination confers long-lasting protection against H5N1 avian influenza in 23 zoo bird species

doi: 10.1038/s41467-025-64301-5

Figure Lengend Snippet: a Genome maps of VSV and VSVΔG(H5) vector. VSV contains five genes (N, P, M, G, and L). In VSVΔG(H5), the G gene is replaced by the HA gene of A/Dalmatian Pelican/Bern/1/2022 (H5N1) encoding either a polybasic (pb) or monobasic (mb) cleavage site. b Infectious virus yield on BHK-G43 helper cells infected with VSVΔG(H5 mb ) at m.o.i. 0.05 f.f.u./cell. At 20 h p.i., virus particles were concentrated from 200 mL of cell culture supernatant by ultracentrifugation (UC) and resuspended in 20 mL PBS. Infectious titers before and after concentration were determined on BHK-21 cells. The infectious titers of n = 12 vaccine batches are shown (blue triangles). The height of the bars indicates the geometric mean value. The error bars show the 95% confidence interval. Statistical significance was tested by the unpaired, two-sided Student’s t test (df = 22; t = 4.732). c Western blot of VSVΔG(H5 pb ) and VSVΔG(H5 mb ) particles after ultracentrifugation. Viral proteins were separated by SDS-PAGE under reducing conditions, stained with colloidal Coomassie (left panel), or immunostained with chicken polyclonal anti-H5 serum. The relative molecular mass (in kDa) according to the migration of molecular weight markers is indicated on left hand side. A representative experiment out of two performed is shown. d Surface H5 antigen was detected with bovine α-H5 serum. VSV matrix (M) protein was stained in permeabilized cells using mAb 23H12 (α-VSV M). VSV*ΔG-infected cells visualized by GFP. Nuclei stained with DAPI. Scale bar = 20 μm. A representative experiment out of two performed is shown. e Multicycle virus replication on MDCK cells. Cells were infected with the indicated viruses (m.o.i. = 0.0001) and cell culture supernatant sampled at 1, 24, and 48 h p.i. Infectious titers were determined on BHK-21 cells (mean ± SD of 3 infection experiments). The detection limit (25 f.f.u. mL -1 ) is indicated (dashed line). The two-way ANOVA with Tukey’s multiple comparison test compared VSVΔG(H5 P :N1 P :GFP) to VSVΔG(H5 pb ) and VSVΔG(H5 mb ). P values are indicated in the graph. f Immunofluorescence analysis of MDCK cells at 20 h p.i. with the indicated viruses (m.o.i. = 0.02). Cells infected with VSVΔG(H5 P :N1 P :GFP) were detected by GFP fluorescence. Cells infected with either VSVΔG(H5 mb ) or VSVΔG(H5 pb ) were detected by indirect immunofluorescence using a monoclonal antibody directed to the VSV M protein. Cell nuclei were stained with DAPI. The bar is equivalent to 100 μm. A representative experiment out of three performed is shown. Source data are provided as a Source Data file.

Article Snippet: The cells were washed with PBS, permeabilized for 5 min with 0.25% (vol/vol) of Triton X-100 in PBS and then incubated for 1 h with a monoclonal antibody directed to VSV matrix (M) protein (mAb 23H12, KeraFast, Boston, MA, cat. no. EB0011), diluted 1:25 in PBS.

Techniques: Plasmid Preparation, Virus, Infection, Cell Culture, Concentration Assay, Western Blot, SDS Page, Staining, Migration, Molecular Weight, Comparison, Immunofluorescence, Fluorescence