Six-week-old male BALB/c mice were obtained from the Jackson laboratory (Bar Harbor, ME). Animals were housed in a temperature-controlled, 12-hour light-cycled facility at the Animal Research Center of The University of Texas at Austin. Mice were given free access to standard rodent chow (Harlan Teklad, Indianapolis, IN) and tap water. Animals were anesthetized by a single intraperitoneal injection of a 3.9:1 mixture of ketamine (100 mg/ml, Putney, Portland, ME) and xylazine (100 mg/ml, Sigma-Aldrich, St. Louis, MO). Once deep plane anesthesia was achieved, animals were immunized with 2000 CEID50 (Chicken Embryo Infectious Dose 50% endpoint) of influenza A virus, A/Puerto Rico/8-9VMC3/1934 (H1N1) (BEI Resources, Manassas, VA) by various routes. Intramuscular injection involved direct injection of virus diluted in saline into each gastrocnemius muscle located on the hindlimb (50 μl per muscle). Nasal immunization was performed by slowly dripping virus diluted in saline into each nostril (10 μl per nostril) using a standard micropipette (Gilson, Middleton, WI). For SL immunization, the SL epithelium was swabbed dry and 20 μl of saline was added to act as an adhesive for the film. Each film was subsequently placed under the tongue using sterile forceps. For BU immunization, the upper part of the check pouch was swabbed dry and 10 μl of saline was added to each cheek to act as an adhesive for the film. Half of each film was placed on the upper part of each cheek pouch using sterile forceps. For both SL and BU doses, dissolution time was complete within 5 min without the animals swallowing or chewing the film. Animals given the vaccine by the nasal, SL, or BU routes were maintained in an upright position for 30 min after immunization to minimize choking and accidental swallowing of the vaccine.

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