(A) Immunization schedule and mice survival after IN plus IM immunization with α-PA vaccine (3×107 CFU) (IN+IM, n = 7), IM immunization with α-PA vaccine (3×107 CFU, n = 8), IN immunization with α-PA vaccine (3×107 CFU) (IN, n = 5), or saline administration (IM, n = 8; IN, n = 8) and challenge with PAO1 ExoU+ (4×105 CFU). *P<0.05 (log-rank test). Images show lungs and livers collected from saline mice which succumbed to infection 16 h after challenge and from random vaccinated mice, sacrificed one week after challenge. Dashed squares indicate pulmonary hemorrhages and pale zones in livers. (B) Immunization schedule and mice survival after IN plus IM immunization with α-PA vaccine (3×107 CFU) (IN+IM, n = 9), IN immunization with α-PA vaccine (3×107 CFU) (IN, n = 9), or saline administration (IN+IM, n = 9) and challenge with PA14 (1×106 CFU). *P<0.05 (log-rank test). (C) Immunization schedule and mice survival after IN plus IM immunization with α-PA vaccine (3×107 CFU) (IN+IM, n = 9), IM immunization with α-PA vaccine (3×107 CFU, n = 9), IN immunization with α-PA vaccine (3×107 CFU) (IN, n = 9), or saline administration (IN+IM, n = 8; IM, n = 9; IN, n = 9) and challenge with PA14 (1×106 CFU). *P<0.05 (log-rank test). Images show lungs and livers collected 12 days after challenge from random vaccinated mice. (D) Immunization schedule and bacterial loads in lungs and livers of mice after IN plus IM immunization with α-PA vaccine (3×107 CFU) (IN+IM, n = 8), IM immunization with α-PA vaccine (3×107 CFU, n = 10), IN immunization with α-PA vaccine (3×107 CFU) (IN, n = 8), or saline administration (IN+IM, n = 8; IN, n = 8) and challenge with PA14 (1×106 CFU). *P<0.05 (Kruskal-Wallis and Mann-Whitney U test were used for 3- and 2-group comparison, respectively).