Evaluation of flagella and flagellin of Pseudomonas aeruginosa as vaccines

Abstract

Pseudomonas aeruginosa is a serious pathogen in hospitalized, immunocompromised, and cystic fibrosis (CF) patients. P. aeruginosa is motile via a single polar flagellum made of polymerized flagellin proteins differentiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, but whether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been addressed, nor has the question of whether such antibodies might negatively impact Toll-like receptor 5 (TLR5) activation, an important component of innate immunity to P. aeruginosa. We compared immunization with flagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using a mouse pneumonia model. Antibodies to flagella were superior to antibodies to flagellin at inhibiting motility, promoting opsonic killing, and mediating protection against P. aeruginosa pneumonia in mice. Protection against the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinical isolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purified flagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizing antibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibited TLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunity to P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity due to TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additional components beyond type a and type b proteins from prototype strains.

FIG. 1.

Assessment of inhibition of motility of P. aeruginosa strains PAK and PA01 with antibodies raised to either flagellin or flagella. The wells containing motility agar were inoculated with the indicated P. aeruginosa strain: PA01 (type b flagella) or PAK (type a flagella) or the corresponding strain lacking the fliC gene. Specific antisera were also added to the wells at the indicated dilution.

FIG. 2.

Phagocyte-dependent killing activity of antibody to P. aeruginosa flagellin or flagella against P. aeruginosa strains PAK and PA01. (A) Anti-flagellin type a serum. (B) Anti-flagellin type b serum. (C) Anti-flagellum type a serum. (D) Anti-flagellum type b serum. Bars represent means of duplicate-quadruplicate determinations, and error bars represent SEM. Based on nonlinear regression determination of the titer of antibody needed to mediate killing of 50% of the target bacteria, comparisons showed results to be significantly (P < 0.05) different.

FIG. 3.

Survival of mice passively immunized with antibody to monomeric flagellin or polymeric flagella. (A and B) Mice immunized with antibodies to monomeric flagellin and challenged with P. aeruginosa type a strain PAK (≈4.75 × 10⁷ CFU/mouse; log rank test, anti-flagellin type a versus NRS, P = 0.88; median survival, 48 h, either group) or type b PA01ExoU⁺ (7 × 10⁵ CFU/mouse; log rank test, anti-flagellin type b versus NRS, P = 0.35; median survival, 48 h, both groups). (C to F) Mice passively immunized with antibody to polymeric flagella and challenged with P. aeruginosa strain PAK (≈3.5 × 10⁷ CFU/mouse; log-rank test, antibody to type a flagella versus NRS, P < 0.0001; antibody to type a flagella versus antibody to type b flagella, P < 0.0001; antibody to type b flagella versus NRS, P = 0.06; median survival: anti-flagellum type a, undefined; anti-flagellum type b, 36 h; NRS, 48 h) (C), with P. aeruginosa strain PA01ExoU⁺ (6 × 10⁶ CFU/mouse; log rank test, antibody to type b flagella versus NRS, P < 0.0001; antibody to type b flagella versus antibody to type a flagella, P = 0.079; antibody to type a flagella versus NRS, P < 0.0005; median survival, antibody to type b flagella, undefined; antibody to type a flagella, 120 h; NRS, 36 h) (D), P. aeruginosa type a strain CF6 (1.6 × 10⁷ CFU/mouse; log-rank test, antibody to type a flagella versus NRS, P = 0.0028; antibody to type a flagella versus antibody to type b flagella, P = 0.18; antibody to type b flagella versus NRS, P = 0.13); median survival, antibody to type a flagella, 114 h; antibody to type b flagella, 54 h; NRS, 48 h) (E), or P. aeruginosa type b strain CF20 (1.37 × 10⁷ CFU/mouse; log-rank test, antibody to type b flagella versus NRS, P = 0.025; antibody to type b flagella versus antibody to type a flagella, P = 0.88; antibody to type a flagella versus NRS, P = 0.009); median survival, antibody to type b flagella, 30 h; antibody to type a flagella, 36 h; NRS, 24) (F).

FIG. 4.

Survival of mice after active vaccination with flagella. (A) Mice challenged with P. aeruginosa PAK (≈2.2 × 10⁷ CFU/mouse) (log rank test, type a flagella versus type b flagella, P < 0.0001; type a flagella versus BSA, P = 0.0016; type a flagella versus LPS, P < 0.0001; median survival, type a flagella, undefined; type b flagella, 42 h; BSA, 36 h, LPS, 48 h). (B) Mice challenged with P. aeruginosa PA01ExoU⁺ (≈8 × 10⁵ CFU/mouse) (log rank, type b flagella versus type a flagella, BSA, or LPS, P < 0.0001; median survival, type b flagella, undefined; type a flagella, 36 h; BSA, 24 h; LPS, 24 h). (C) Mice challenged with P. aeruginosa CF6 (type a) (≈2.56 × 10⁷ CFU/mouse) (log rank, type a flagella versus type b flagella, P < 0.0007; type a flagella versus BSA, P = 0.0305; type a flagella versus LPS, P = 0.094; median survival, type a flagella, 102 h; type b flagella, BSA, and LPS, 36 h). (D) Mice challenged with P. aeruginosa CF20 (type b) (≈1.25 × 10⁷ CFU/mouse) (log-rank, type b flagella versus type a flagella, P = 0.013; type b flagella versus BSA, P = 0.089; type b flagella versus LPS, P = 0.0133; median survival, type b flagella, 54 h; type a flagella, BSA, and LPS, 36 h). BSA- and LPS-immunized mice served as the controls in all experiments. Results are represented in Kaplan-Meier survival curves and analyzed by the log rank test. “n” refers to the number of animals.

FIG. 5.

Activation of TLR5 by P. aeruginosa flagellin or flagella. Addition of the indicated amount of purified protein induced the production of luciferase initiated by TLR5-dependent activation of NF-κB and binding of nuclear translocated activated NF-κB fragments to cognate sites in the luc gene promoter. The points are the means of quadruplicate measurements, and error bars represent SEM.

FIG. 6.

Effect of antibody to flagella or flagellin on TLR5 activation. (A) Effect of antibody to type a flagella or flagellin on the activation of TLR5 by type a flagella. (B) Effect of antibody to type b flagella or flagellin on the activation of TLR5 by type b flagella. (C) Effect of antibody to type a flagella or flagellin on the activation of TLR5 by type a flagellin. (D) Effect of antibody to type b flagella or flagellin on the activation of TLR5 by type b flagellin. The points are the results of triplicate determinations, and error bars represent SEM. Normal rabbit serum (NRS) was also included for comparison.

FIG. 7.

Effects of antibody to flagella or flagellin on activation of TLR5 by whole bacterial cells. (A) Luciferase production due to activation of TLR5 by P. aeruginosa strain PAK, PA01, PAKΔfliC, or PA01ΔfliC. Both wild-type strains induced TLR5 activity, whereas the strains lacking flagella were devoid of agonist activity. (B) Activation of TLR5 by P. aeruginosa strain PAK bacteria and inhibition by antisera raised to either type a flagella or flagellin at a 1:50 dilution. (C) Activation of TLR5 by P. aeruginosa strain PA01 bacteria and inhibition by antibody to type b flagella or flagellin at a 1:50 dilution. NRS was used as a control. The points are means of triplicate determinations, and error bars represent SEM.