Fig 1.
α-PA vaccine generates cross-reactive antibodies and heterogeneous protection against PA-pneumonia.
(A) Immunization schedule and antibody titers against PAO1 in vaccinated (n = 7) and control (n = 7) mice after one or two IN immunizations with α-PA vaccine (2×108 CFU), or saline administration, respectively. (B) IgG titers against different live and Hk PA strains in vaccinated (n = 6–7) and control (n = 5–7) mice after two IN immunizations with α-PA vaccine (2×108 CFU), or saline administration, respectively. (C) Immunization schedule and antibody titers against PAO1 in vaccinated (n = 5) and control (n = 6–7) mice after three IN immunizations with α-PA vaccine (2×108 CFU), or saline administration, respectively. (A-C) S, saline; D, day. (A, B) *P<0.05 (Kruskal-Wallis test), compared with saline and between the indicated groups. (B) #P<0.05 (Mann-Whitney U test), compared with saline group. (C) *P<0.05 (Mann-Whitney U test), compared with saline group. (D) Immunization schedule, mice survival and assessment of murine disease severity score (MDSS) after IN administration of α-PA vaccine (2×108 CFU), or saline and IN challenge with PAO1 ExoU+ (7×105 CFU, n = 7), PA14 (1×106 CFU, n = 8), ST235 (3×107 CFU, n = 7) and PAO1 (2×106 CFU, n = 7–8). (E) Mice survival after IN challenge with different doses of PAO1 (n = 3–5 mice/group). (F) Immunization schedule and mice survival after IN administration of α-PA vaccine (2×108 CFU, n = 6), or saline (n = 7) and IN challenge with PA14 (1×106 CFU). (G) Mice survival after IN immunization with α-PA vaccine (2×108 CFU, n = 8), inactivated PAO1 (2×108 CFU, n = 8) or saline administration (n = 15) and IN challenge with PA14 (1×106 CFU), according to schedule (D). (D, F and G) *P<0.05 (log-rank test), compared with saline group. (D) #P<0.05 (Mann-Whitney U test). (G) #P<0.05 (log-rank test), compared with mice administered inactivated PAO1.
Fig 2.
Production of memory CD4+ T cells in vaccinated mice.
(A) Mice (n = 6/group) were immunized with α-PA vaccine (2×108 CFU) or administered saline, according to the schedule; then spleens were collected. (B) Percentage of spleen effector memory (CD44+ CD62L-), central memory (CD44+ CD62L+) and naïve (CD44- CD62L+) CD4+ T cells. Contour plots correspond to a representative example of CD4-gated T cells of the analyzed samples. Analysis regions were set according to FMO and isotype control-stained samples. Numbers inside plot regions represent means ± SD of the frequency of cells due to respective cell staining. Bars represent mean ± SD of data. *P<0.05 (t-test).
Fig 3.
Increased production of IL-17A by CD4+ T cells of vaccinated mice.
(A, B) Mice (n = 6/group) were immunized with α-PA vaccine (2×108 CFU) or administered saline, according to the schedule; then spleens and lungs were collected. In (B), mice (n = 6/group) were infected with PA14 (1×106 CFU), 9 h before the spleens and lungs were obtained. (C, D) Representative examples and percentage of CD4-gated T cells expressing IL-17A obtained from spleens (C) and lungs (D) of infected and non-infected mice, detected by intracellular staining after stimulation with PMA/ionomycin. Analysis regions were set according to FMO and isotype control-stained samples. Numbers inside dot plot regions represent means ± SD of the frequency of cells due to respective cytokine staining. Bars represent mean ± SD of data. *P<0.05, **P<0.01 (t-test).
Fig 4.
IM immunization with α-PA vaccine generates systemic and cross-reactive antibodies but reduced protection against pneumonia.
(A) Immunization schedule for (B) and (C). (B) Antibody titers against PAO1 in vaccinated (n = 6) and control (n = 6) mice after IM immunization with α-PA vaccine (3×107 CFU), or saline administration, respectively. (C) IgG titers against different live and Hk PA strains in vaccinated (n = 6) and control (n = 6) mice after IM immunization with α-PA vaccine (3×107 CFU), or saline administration, respectively. (B, C) S, saline; D, day. *P<0.05 (Mann-Whitney U test), compared with saline group. (D) Immunization schedule and mice survival after IM immunization with α-PA vaccine (3×107 CFU, n = 8), or saline administration (n = 9) and challenge with PAO1 ExoU+ (1×105 CFU). (E) Immunization schedule and mice survival after IM immunization with α-PA vaccine (3×107 CFU, n = 8), or saline administration (n = 8) and challenge with PA14 (9×105 CFU). (F) Immunization schedule and mice survival after IM immunization with α-PA vaccine (3×107 CFU, n = 9), or saline administration (n = 9) and challenge with ST235 (5×106 CFU). (D-F) *P<0.05 (log-rank test).
Fig 5.
Combination of IN plus IM immunization with α-PA vaccine and protection against pneumonia.
(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).
Fig 6.
Lung histopathology caused by PA-pneumonia in vaccinated and naive mice.
(A) Immunization and lung collection schedule for (C). (B) Immunization, challenge and lung collection schedule for (D). (C) Representative photomicrographs of histological sections of lungs obtained from mice after IN (IN) immunization with α-PA vaccine (3×107 CFU) (n = 3), IN plus IM (IN+IM) immunization with α-PA vaccine (3×107 CFU) (n = 3) and saline administration (IN+IM) (n = 3). (C) Arrows show perivascular and periarteriolar foci of inflammatory infiltrate of the mononuclear type in the parenchyma. (D) Representative photomicrographs of histological sections of lungs obtained from mice after IN (IN) immunization with α-PA vaccine (3×107 CFU) (n = 6), IN plus IM (IN+IM) immunization with α-PA vaccine (3×107 CFU) (n = 5), saline administration (IN+IM) (n = 5) and challenge with PA14 (1×106 CFU). (D) Arrows show a severe inflammatory infiltrate of the polymorphonuclear type dispersed throughout the pulmonary parenchyma. Arrowheads show polymorphonuclear infiltrate filling most of the alveoli (saline), areas of scarce inflammatory mononuclear infiltrate (IN-immunized mice) and areas of inflammatory infiltrate of the polymorphonuclear type leukocyte with intrabronchial location (IN+IM-immunized mice), respectively. (C, D) The columns show images with ×25 (left), ×100 (center left), ×200 (center right) and ×400 (right) magnifications.
Fig 7.
Live α-PA vaccine generates mucosal antibody responses and functional IgG in blood.
(A) Immunization schedule and IgA titers in BAL 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; IM, n = 7), IN immunization with α-PA vaccine (3×107 CFU; IN, n = 8), or saline administration (IN+IM, n = 9; IM, n = 9; IN, n = 9). Titers were normalized respective to 50 μL. (B) Immunization schedule and IgA titers in VLF 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; IM, n = 7), IN immunization with α-PA vaccine (3×107 CFU; IN, n = 8), or saline administration (IN+IM, n = 8; IM, n = 8; IN, n = 8). (A, B) S, saline. *P<0.05 (Kruskal-Wallis test), between the indicated groups. (C) Immunization schedule and in vitro OPKA assay, representing percent killing of PAO1 (2×104 CFU) and PA14 (2×104 CFU) by α-PA serum (or naïve serum) in the presence of human PMNs. α-PA and naïve sera were obtained from the blood of mice after IN immunization with α-PA vaccine (3×107−2×108 CFU, n = 6) or saline administration (n = 6), respectively. *P<0.05 (Mann-Whitney U test).