To study
the long-term immune
responses, we evaluated the Aic virus and hrHA3-specific antibody
levels at 3- and 6-months postboosting immunization and then challenged
the mice (n = 3–4) with 2 × LD50 of the Philippines virus. Here we included the H3+CpG group. After
monitoring the mouse body weight changes daily for 6 days, we euthanized
the mice, collected the mucosal BALF lymphocytes, and studied the
local cellular responses by flow cytometry.53 (link) Briefly, after antigen restimulation for 5 h in the presence of
a Golgi stopper (BD Biosciences, 2 μg mL–1), the cells were stained by CD4-PE-Cy7 and CD8-PE-Cy5 antibodies
(BD Biosciences). After fixation and permeabilization, intracellular
staining with IFN-γ-BV711 (BioLegend) was performed. IFN-γ-secreting
T lymphocytes were recorded by flow cytometry (BD LSRFortessa) and
analyzed by FlowJo software.
the long-term immune
responses, we evaluated the Aic virus and hrHA3-specific antibody
levels at 3- and 6-months postboosting immunization and then challenged
the mice (n = 3–4) with 2 × LD50 of the Philippines virus. Here we included the H3+CpG group. After
monitoring the mouse body weight changes daily for 6 days, we euthanized
the mice, collected the mucosal BALF lymphocytes, and studied the
local cellular responses by flow cytometry.53 (link) Briefly, after antigen restimulation for 5 h in the presence of
a Golgi stopper (BD Biosciences, 2 μg mL–1), the cells were stained by CD4-PE-Cy7 and CD8-PE-Cy5 antibodies
(BD Biosciences). After fixation and permeabilization, intracellular
staining with IFN-γ-BV711 (BioLegend) was performed. IFN-γ-secreting
T lymphocytes were recorded by flow cytometry (BD LSRFortessa) and
analyzed by FlowJo software.
