Antibiotic-treated mice fail to induce acquired immunity to influenza virus infection. C57BL/6 mice were given ampicillin (1 g/L), vancomycin (500 mg/L), neomycin sulfate (1 g/L), and metronidazole (1 g/L) in drinking water for 4 wk before PR8 virus infection (10 pfu per mouse). Two weeks later, serum and nasal wash were collected and Ag-specific antibody titers were measured (A), and T-cells were isolated from spleen and restimulated with flu virion or NP peptide for 72 h, and IFN-γ production from CD4 T cells (B) and CD8 T cells (C) was measured. (D) Lymphocytes were collected from the lung of infected animals at 14 d postinfection and stained with flu-specific tetramer. (E) The lung washes of flu-infected mice were harvested at 9 d postinfection, and viral titers were measured by plaque assay. *P < 0.05 and ***P < 0.001 vs. water-fed group. Data represent the mean ± SD. Similar results were obtained from three separate experiments.