A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology

Fig. 1. Kinetics of HPIV-3 replication in intranasally inoculated AG129 mice. a Setup of the study. AG129 mice were intranasally inoculated with 1.5 × 106 TCID50 of HPIV-3. Lung samples and NPLT-washes were collected at various time points post-infection (p.i.). b Infectious virus titers are expressed as log10 TCID50 per milligram of lung sample. c Infectious virus titers are expressed as log10 TCID50 per milliliter of NPLT-wash. d Weight change at various time points post-infection is presented as a percentage of the body weight at the time of inoculation. e Cumulative severity scores of lungs of all infected mice. Individual data and median values (indicated by bars) are presented in all graphs. LLOD presents the lower limit of detection. Data are from one experiment with four mice per group. Source data are provided as a Source Data file. a was designed with BioRender.

Fig. 2. Tropism of HPIV-3 to ciliated cells and club cells of the bronchiolar epithelium of AG129 mice. Confocal images of 5 µm sections through lung samples. Multiplex fluorescence with RNAscope and IHC. The name of the mouse gene or viral RNA that is the target for an RNAscope probe, yielding puncta, is in italics. The name of the protein that is the target for an antibody, yielding an immunoreactive (IR) signal, is in roman. DAPI serves as a nuclear stain. a–c Uninfected, d, e 1 d.p.i., f 10 d.p.i., g–i 3 d.p.i., j–l 5 d.p.i. a Transverse section through a bronchiole. The lumen of the bronchiole (the black space at the center) is devoid of cells. Alveoli of the lung parenchyma surround the bronchiole. Foxj1 is a marker for ciliated cells and Scgb1a1 for club cells in the bronchiolar epithelium. Sftpc is a marker for type II pneumocytes in the alveolar wall. b, c As expected, there are no puncta for the HPIV-3-HN probe and the HPIV-3-NP probe in uninfected mice. d The Krt8-IR signal labels most epithelial cells in the bronchiolar epithelium. A fraction of the cells in the bronchiolar epithelium harbor HPIV-3-HN puncta and HPIV-3-NP puncta, colocalizing within the same cells. There are…

Fig. 3. Prophylactic oral treatment with GS-441524 of AG129 mice intranasally inoculated with HPIV3 reduces viral loads and lung pathology. a Setup of the study. AG129 mice were orally treated twice daily with either vehicle or 50 mg/kg GS-441524 from day −1 p.i. to day 2 p.i., and intranasally inoculated with 1.5 × 106 TCID50 of HPIV-3 at day 0 p.i. Lung samples were collected at day 3 p.i. to measure viral loads. b Infectious virus titers are expressed as log10 TCID50 per milligram of lung sample. LLOD presents the lower limit of detection. c HPIV-3 RNA levels are expressed as log10 TCID50 equivalent per milligram of lung sample. LLOQ presents the lower limit of quantification. b, c Data are from two independent experiments, each with 6 mice per group. Data were analyzed by the two-tailed Mann–Whitney U test. ****p < 0.0001. d Setup of the study. AG129 mice were orally treated twice daily with vehicle or 50 mg/kg GS-441524 from day −1 p.i. to day 5 p.i., and intranasally inoculated with 1.5 × 106 TCID50 of HPIV-3 at day 0 p.i. Lungs were collected at day 6 p.i. for lung histopathology assessment. e Weight change at day 6 p.i. is presented as a percentage of the body weight at day −1…

Fig. 4. Visualizing HPIV-3 RNA species in vehicle-treated vs. GS-441524-treated AG129 mice. Confocal images of sections through lung samples. Multiplex fluorescence with double or triple RNAscope. The name of a viral RNA species that is a target for an RNAscope probe, yielding puncta, is in italics. HPIV-3-NP-antigenome puncta visualize positive-sense viral RNA species. DAPI serves as nuclear stain. a Uninfected, b–f vehicle-treated, g–i GS-441524-treated. a Transverse section through two adjacent bronchioles. The lumen of the bronchioles (the black spaces at the center) is devoid of cells. Foxj1 is a marker for ciliated cells. As expected, there are no puncta for the HPIV-3-HN probe and for the HPIV-3-NP-antigenome probe in this uninfected mouse. b HPIV-3-HN puncta diffusely fill cells in the bronchiolar epithelium, and HPIV-3-NP-antigenome puncta are perinuclear. c An orthogonal projection of a z-stack of confocal slices of the region in (b) indicated with an asterisk. A cell at the center harbors both Foxj1 puncta and HPIV-3-NP-antigenome puncta; these puncta appear yellow in overlay. d Scgb1a1 is a marker for club cells in the bronchiolar epithelium. A great many cells in the…

Fig. 5. Intranasal treatment with GS-441524 of intranasally inoculated AG129 mice reduces viral loads in the lung and NPLT-washes and lung pathology. a Setup of the study. AG129 mice were intranasally treated once daily with either vehicle or GS-441524 (40 µL/mouse of a 30 mg/mL solution) from day −1 p.i. to day 2 p.i., and intranasally inoculated with 1.5 × 106 TCID50 of HPIV-3 at day 0 p.i. Lung samples and NPLT-washes were collected at day 3 p.i. to measure viral loads. b Infectious virus titers are expressed as log10 TCID50 per milligram of lung sample. c HPIV-3 RNA levels are expressed as log10 TCID50 equivalent per milligram of lung sample. d Infectious virus titers are expressed as log10 TCID50 per milliliter of NPLT-wash. e HPIV-3 RNA levels are expressed as log10 TCID50 equivalent per milliliter of NPLT-wash. b–e LLOD presents the lower limit of detection. LLOQ presents the lower limit of quantification. Data are from two independent experiments with ten mice per group. Data were analyzed by the two-tailed Mann–Whitney U test. *p = 0.0312, **p = 0.0071, ****p < 0.0001. f Setup of the study. AG129 mice were intranasally treated once daily with either vehicle or GS-441524 (40…

Fig. 6. A single intranasal prophylactic dose of GS-441524 in AG129 mice intranasally inoculated with HPIV-3 reduces viral loads in the lung. a Setup of the study. AG129 mice were administrated intranasally with a single dose of GS-441524 (40 µL/mouse of a 30 mg/mL solution) at 6 or 1 h pre-infection, and then intranasally inoculated with 1.5 × 106 TCID50 of HPIV-3. Lung samples were collected at day 3 p.i. to measure viral loads. b Infectious virus titers are expressed as log10 TCID50 per milligram of lung sample. LLOD presents the lower limit of detection. c HPIV-3 RNA levels are expressed as log10 TCID50 equivalent per milligram of lung sample. Data are from one experiment with five mice per group. Data were analyzed with the Kruskal–Wallis with Dunn’s multiple comparisons test. *p = 0.0390 (b), *p = 0.0392 (c), **p = 0.0060. Individual data and median values (indicated by bars) are presented in all graphs. Source data are provided as a Source Data file. a was designed with BioRender.