Sublingual and Intranasal RSV Vaccination

Female BALB/c mice were purchased from Charles River Laboratories Inc. (Yokohama, Japan) and kept under specific pathogen-free conditions. For the immunization, 6 to 8-week-old mice were inoculated twice on day 0 and day 14 with 20 µg of purified G protein fragment via the sublingual (s.l.) or intranasal (i.n.) route. For s.l. immunization, mice were anesthetized by i.p. injection of ketamine and recombinant RSV G fragment with or without cholera toxin (2 µg) in 15 µl were delivered underneath the tongue. For i.n. immunization, mice were lightly anesthetized by isoflurane inhalation, and 50 µl of vaccine or PBS were applied to the left nostril. FI-RSV (1×10⁵ PFU in 50 µl) with aluminum hydroxide (Sigma-Aldrich, Seoul, Korea) was administered once through the foot pad of anesthetized mice. As a positive control, live RSV (1×10⁵ PFU) was i.n. delivered once. Three to four weeks after the last immunization, the mice were challenged i.n. with 1×10⁶ or 2×10⁶ PFU of live RSV A2, if necessary. All animal studies were performed with approval of our Institutional Animal Care and Use Committee (Approval No. 2010-9-4).