Influenza B Virus Challenge Assay

To analyze the VLP vaccine-induced changes in immune cell populations, flow cytometry was performed. Briefly, lung tissues were harvested after 4 dpi with the influenza B virus challenge infection. After homogenizing the tissues, single cell populations of lung cells were prepared by Percoll density gradient as described previously [31 (link)]. Cells were stimulated with inactivated B/Colorado/06/2017 virus antigen (0.4 ug/mL) for 2 h at 37 °C. CD4+ T cell, CD8+ T cell, B cell, and germinal center (GC) B cell populations were measured by staining the cells with fluorophore-conjugated CD3, CD4, CD8, B220, GL7, CD19, and IgD antibodies (BD Biosciences, San Diego, CA, USA). Stained cells were acquired on C6 flow cytometer and analyzed using C6 Analysis Software (BD Bioscience, San Diego, CA, USA).