Figure 1.
Schematic of animal study. (A) Animal study outline: Eighty-eight BALB/c mice (n = 11) were intramuscularly vaccinated with COBRA or wild-type HA VLP vaccines formulated with AddaVax adjuvant at weeks 0, 4, and 8. At weeks 6 and 10 post vaccination, blood was collected, and the sera were separated for analysis. At week 12, all mice were inoculated with 5 × 104 PFU of A/California/07/2009 H1N1 virus intranasally, lung tissues (n = 3/group) were harvested on days 3 and 6 post infection and evaluated for histopathology and virus titration. (B) Body weight loss of mice post infection: The mice were observed for clinical signs for 14 days, and their body weight was recorded daily post infection. The dotted line indicates 80% of their body weights on D0 post infection. (C) Survival cure after infection with A/California/07/2009 virus. Another 64 DBA/2J mice were intramuscularly vaccinated with COBRA or wild-type rHA vaccines formulated with AddaVax adjuvant using the same vaccination regimen mentioned above. At week 12, all mice were intranasally infected with 8.75 × 106 PFU of A/Brisbane/02/2018 H1N1 virus. (D) Body weight loss curve of DBA/2J mice after infection with A/Brisbane/02/2018 H1N1 virus. (E) Survival cure after infection with A/Brisbane/02/2018 virus.
Figure 1.
Schematic of animal study. (A) Animal study outline: Eighty-eight BALB/c mice (n = 11) were intramuscularly vaccinated with COBRA or wild-type HA VLP vaccines formulated with AddaVax adjuvant at weeks 0, 4, and 8. At weeks 6 and 10 post vaccination, blood was collected, and the sera were separated for analysis. At week 12, all mice were inoculated with 5 × 104 PFU of A/California/07/2009 H1N1 virus intranasally, lung tissues (n = 3/group) were harvested on days 3 and 6 post infection and evaluated for histopathology and virus titration. (B) Body weight loss of mice post infection: The mice were observed for clinical signs for 14 days, and their body weight was recorded daily post infection. The dotted line indicates 80% of their body weights on D0 post infection. (C) Survival cure after infection with A/California/07/2009 virus. Another 64 DBA/2J mice were intramuscularly vaccinated with COBRA or wild-type rHA vaccines formulated with AddaVax adjuvant using the same vaccination regimen mentioned above. At week 12, all mice were intranasally infected with 8.75 × 106 PFU of A/Brisbane/02/2018 H1N1 virus. (D) Body weight loss curve of DBA/2J mice after infection with A/Brisbane/02/2018 H1N1 virus. (E) Survival cure after infection with A/Brisbane/02/2018 virus.
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Figure 6.
Lung injury in mice after A/California/09/2007 virus infection. Three mice from each group were euthanized on day 3 (A) and day 6 post infection (B). The left lungs were inflated with 10% buffered formalin and then embedded into paraffin blocks. H&E staining was performed on 3 sectional slides for each mouse lung sample. Neutrophils in alveolar space, neutrophils in interstitial space, proteinaceous debris filling the airspaces, and alveolar septal thickening were assessed. Each parameter was evaluated in 10 random fields under 40× magnification (scale bar indicated 50 µm), and then, the total injury scores of 10 fields were determined. Scoring system: Neutrophils in alveolar space (indicated with orange arrows): 0 = none, 1 = 1–5, 2 = >5; neutrophils in interstitial space (indicated with green arrows): 0 = none, 1 = 1–5, 2 = >5; proteinaceous debris filling the airspaces (indicated with yellow arrows): 0 = none, 1 = 1, 2 = >1; alveolar septal thickening (indicated with blue arrows): 0 = <2×, 1 = 2×–4×, 2 = >4×. (C) Normal mouse left lung (no vaccination or infection). (D–K) Representative images of alveolar septal thickening on day 6 post infection in mice vaccinated with (D) Bris/18, (E) Y2, (F) Y4, (G) CA/09, (H) P1, (I) X6, (J) Bris/07, and (K) PBS. The data are presented as absolute mean plus SEM. A one-way ANOVA was used to analyze the statistical differences of the lung injury scores using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
Figure 6.
Lung injury in mice after A/California/09/2007 virus infection. Three mice from each group were euthanized on day 3 (A) and day 6 post infection (B). The left lungs were inflated with 10% buffered formalin and then embedded into paraffin blocks. H&E staining was performed on 3 sectional slides for each mouse lung sample. Neutrophils in alveolar space, neutrophils in interstitial space, proteinaceous debris filling the airspaces, and alveolar septal thickening were assessed. Each parameter was evaluated in 10 random fields under 40× magnification (scale bar indicated 50 µm), and then, the total injury scores of 10 fields were determined. Scoring system: Neutrophils in alveolar space (indicated with orange arrows): 0 = none, 1 = 1–5, 2 = >5; neutrophils in interstitial space (indicated with green arrows): 0 = none, 1 = 1–5, 2 = >5; proteinaceous debris filling the airspaces (indicated with yellow arrows): 0 = none, 1 = 1, 2 = >1; alveolar septal thickening (indicated with blue arrows): 0 = <2×, 1 = 2×–4×, 2 = >4×. (C) Normal mouse left lung (no vaccination or infection). (D–K) Representative images of alveolar septal thickening on day 6 post infection in mice vaccinated with (D) Bris/18, (E) Y2, (F) Y4, (G) CA/09, (H) P1, (I) X6, (J) Bris/07, and (K) PBS. The data are presented as absolute mean plus SEM. A one-way ANOVA was used to analyze the statistical differences of the lung injury scores using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).
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