Immunization with Bivalent Flagellin Protects Mice against Fatal Pseudomonas aeruginosa Pneumonia

Abstract

Pseudomonas aeruginosa lung infections present a major challenge to healthcare systems worldwide because they are commonly associated with high morbidity and mortality. Here, we demonstrate the protective efficacy of type a and b flagellins (bivalent flagellin) against acute fatal pneumonia in mice. Mice immunized intranasally with a bivalent flagellin vaccine were challenged by different flagellated strains of P. aeruginosa in an acute pneumonia model. Besides the protective effect of the vaccine, we further measured the host innate and cellular immunity responses. The immunized mice in our study were protected against both strains. Remarkably, active immunization with type a or b flagellin significantly improved survival of mice against heterologous strain compared to flagellin a or b antisera. We also showed that after an intranasal challenge by P. aeruginosa strain, neutrophils are recruited to the airways of vaccinated mice, and that the bivalent flagellin vaccine was proved to be protective by the generated CD4⁺IL-17⁺ Th17 cells. In conclusion, bivalent flagellin vaccine can confer protection against different strains of P. aeruginosa in an acute pneumonia mouse model by eliciting effective cellular and humoral immune responses, including increased IL-17 production and improved opsonophagocytic killing.

Figure 1

Survival rates of actively and passively immunized BALB/c mice (n = 12 mice/group) to infection with P. aeruginosa strains. Active and passive immunization against bivalent flagellin (a, b), flagellin a (c, d), and flagellin b (e, f). Kaplan-Meier curves were plotted for mice of the above groups, which were challenged by 2 × 10⁷ CFUs of P. aeruginosa strains PAO1 and PAK and monitored the seven day survival rates. ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 2

The protective role of CD4⁺ T lymphocytes in bivalent flagellin vaccine against P. aeruginosa strains. Bivalent flagellin- (a, b), flagellin a- (c, d), and flagellin b- (e, f) immunized mice (n = 12 mice/group) were treated with either anti-CD8 monoclonal antibody, or anti-CD4 monoclonal antibody, or normal rat IgG and then were challenged with 2 × 10⁷ CFUs of P. aeruginosa strains PAO1 and PAK. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 3

The opsonic killing activity of sera from bivalent flagellin-, flagellin a-, and flagellin b-immunized mice 3 weeks after the final immunization. Values presented as the mean of triplicate technical replicates ± SD. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 4

Assessment of motility inhibition of P. aeruginosa strains PAK (a) and PAO1 (b) by different dilutions of sera from bivalent flagellin-, flagellin a-, or flagellin b-immunized mice. Motility agar plates are prepared with diluted antibodies in each well prior to stabbing each well with fresh overnight bacterial cultures. The mean diameters of P. aeruginosa strains were measured in millimeters (mean + SD). Values presented as mean of triplicate independent experiments ± SD. ^∗P < 0.05 and ^∗∗P < 0.01. NS = not significant. CS = control serum.

Figure 5

Effects of bivalent flagellin immunization on pre and postchallenge levels of serum total IgG against the whole cell of P. aeruginosa PAK (a), PAO1 (b), and mixed flagellins (c). The pre- and postchallenge serum IgG1 (d) and IgG2a (e) titers against mixed flagellins. Values are represented as mean ± SD based on five mice in each group. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 6

Proliferative assay of immune splenic T cells from bivalent flagellin (a, b), flagellin a (c, d), and flagellin b (e, f) stimulated with antigens in vitro for 24 h and 72 h. Values are represented as mean ± SD based on five mice in each group. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 7

Effects of active immunization with bivalent flagellin on IL-17 production of immune splenic T cells from bivalent flagellin (a, b), flagellin a (c, d), and flagellin b (e, f) at 24 h and 48 h after stimulation with flagellin a or flagellin b. Values represented as mean ± SD based on five mice in each group. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 8

The effect of nasal immunization with bivalent flagellin, flagellin a, and flagellin b on the local and systemic spread of P. aeruginosa strains PAK and PAO1. Bacterial load was determined as CFUs (log) in the lung (a), liver (b), spleen (c), and blood (d). Values represented as mean ± SD based on five mice in each group. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 9

Total BALF neutrophils at 6 h (a) and 18 h (b) after infection of immune and nonimmune mice with P. aeruginosa strains PAK and PAO1. The bacterial CFU in BALF at 6 h (a) and 18 h (b) after infection of immune and nonimmune mice with PAK (e) and PAO1 (f). IL-17 level in BALF at 6 h (a) and 18 h (b) after infection of immune and nonimmune mice with PAK (e) and PAO1 (f). Values represented as mean ± SD based on five mice in each group. ^∗P < 0.05 and ^∗∗P < 0.01. NS = nonsignificance.

Figure 10

The role of IL-17 in vaccine based protection against P. aeruginosa strains. Bivalent flagellin- (a, b), flagellin a- (c, d), and flagellin b- (e, f) immunized mice (n = 12 mice/group) were given IL-17 IgG or control IgG for 3 consecutive days prior to challenge with 2 × 10⁷ CFUs of P. aeruginosa strains PAO1 and PAK. ^∗P < 0.05 and ^∗∗P < 0.01.