Evaluation of Flagella and Flagellin of Pseudomonas ...Flagella, obtained as a pellet, were then suspended in a minimum amount of PBS and filtered through a 0.45-m-pore-size Millipore

INFECTION AND IMMUNITY, Feb. 2010, p. 746–755 Vol. 78, No. 20019-9567/10/$12.00 doi:10.1128/IAI.00806-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Evaluation of Flagella and Flagellin of Pseudomonas aeruginosaas Vaccines�

Victoria L. Campodonico,1†* Nicolas J. Llosa,1,2† Martha Grout,1 Gerd Doring,3Tomas Maira-Litran,1 and Gerald B. Pier1

Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts1;Department of Pediatrics, Floating Hospital for Children, Tufts University School of Medicine, Boston, Massachusetts2; and

Institute of Medical Microbiology and Hygiene, University of Tubingen, Tubingen, Germany3

Received 17 July 2009/Returned for modification 2 September 2009/Accepted 1 December 2009

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 differen-tiated into two major serotypes: a and b. Antibodies to flagella delay onset of infection in CF patients, butwhether immunity to polymeric flagella and that to monomeric flagellin are comparable has not been ad-dressed, 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 withflagella and that with flagellin for in vitro effects on motility, opsonic killing, and protective efficacy using amouse 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. Protectionagainst the flagellar type strains PAK and PA01 was maximal, but it was only marginal against motile clinicalisolates from flagellum-immunized CF patients who nonetheless became colonized with P. aeruginosa. Purifiedflagellin was a more potent activator of TLR5 than were flagella and also elicited higher TLR5-neutralizingantibodies than did immunization with flagella. Antibody to type a but not type b flagella or flagellin inhibitedTLR5 activation by whole bacterial cells. Overall, intact flagella appear to be superior for generating immunityto P. aeruginosa, and flagellin monomers might induce antibodies capable of neutralizing innate immunity dueto TLR5 activation, but solid immunity to P. aeruginosa based on flagellar antigens may require additionalcomponents beyond type a and type b proteins from prototype strains.

Pseudomonas aeruginosa is an opportunistic pathogen re-sponsible for a large proportion of ventilator-associated, hos-pital acquired pneumonia and is also a major cause of mor-bidity and mortality in cystic fibrosis (CF) patients. P.aeruginosa is motile via a single polar flagellum that has theadded structural feature of being glycosylated (39). Flagellin isthe primary protein component of the flagellar filament, and itcan be classified into two serotypes, types a and b. Flagellacarry out many functions, such as motility and attachment ofbacteria to host cells, and can also elicit the activation of thehost inflammatory response via Toll-like receptor 5 (TLR5) (6,15, 29, 31). Importantly, promising results in terms of preven-tion of the acquisition of P. aeruginosa infection in CF patientsimmunized with a bivalent type a and b flagellum vaccine havebeen published (12).

Several animal studies have not only demonstrated the im-portance of flagella as a virulence factor in P. aeruginosa butalso validated them, or their flagellin component, as targetantigens for vaccination. In the burned-mouse model of infec-tion, chemically mutagenized or genetically produced flagel-lum-negative strains were less virulent than flagellum-positivestrains (5, 26). It has also been shown with this model thatmotility is necessary for dissemination from the site of infec-

tion, since an intact flagellum structure is essential for deathdue to sepsis (5). In a neonatal model of acute P. aeruginosapulmonary infection, flagella were essential for full virulence(14), although this was not found to be the case for adult micewith pulmonary P. aeruginosa infection (6). In regard to pro-tection mediated by flagella or flagellin, immunization withflagella provided protection against infection and decreasedthe spread to major organs in the burned-mouse model (19). Ina rat model of P. aeruginosa-induced pneumonia, administra-tion of human monoclonal antibodies (MAbs) to flagella pro-vided protection against infection and decreased lung injury(24), and another set of human MAbs provided protection in amurine model of pneumonia during neutropenia (28). A DNAvaccine encoding recombinant type a or type b P. aeruginosaflagellin also induced protective immunity against lethal P.aeruginosa lung infection (33), although this study curiouslyfound better heterologous protection than homologous protec-tion when DNA encoding wild-type flagellin was incorporatedinto the vaccine. A fusion protein of outer membrane proteinF (OprF) residues 311 to 341, mature OprI residues 21 to 83,and flagellins a and b (termed OprF311-341-OprI-flagellins)generated significant immune responses in mice and promotedenhanced clearance of strain PA01 in a pulmonary challengemodel (42). None of these studies directly compared the vac-cine potential of flagellin with that of flagella.

In addition to being highly immunogenic, the flagellin com-ponent of flagella serves as a pathogen-associated molecularpattern (PAMP), activating TLR5 and inducing innate immu-nity in the lung, stimulating a protective inflammatory response

* Corresponding author. Mailing address: Channing Laboratory,181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-2662. Fax:(617) 525-2510. E-mail: [email protected].

† V.L.C. and N.J.L. contributed equally to this work.� Published ahead of print on 7 December 2009.

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that contributes to the eradication of the pathogen (15, 32, 33,35). Instillation of recombinant flagellin into the lungs of miceelicits a significant induction of innate immunity (20), andapplication of flagellin to the cornea of mice or intraperitoneal(i.p.) injection prior to corneal injury and local P. aeruginosainfection protects against pathological destruction of this tissue(22, 23). Finally, overexpression of flagellin monomers en-hances virulence of P. aeruginosa (6).

Of great interest is that the TLR5-binding domain of flagel-lin is not exposed in the intact flagella (36), and thus, flagellinmonomers must be released or extracted from the intact fla-gella to promote TLR5 activation. Therefore, the comparativeTLR5 agonist activity of flagellin, flagella, and even intact P.aeruginosa bacteria has not been evaluated, nor is it clear if theTLR5 activation component of flagellin would be immuno-genic when immunizing with the intact polymeric flagella.

Since P. aeruginosa serotype a and b flagella are conserved,contribute to virulence, stimulate innate immunity, and haveinduced protective efficacy in both animal (19, 24) and human(12) vaccine studies, it is clear that the flagellum or the flagellinmonomer may be a useful target as a vaccine component,particularly as a carrier protein to link to protective carbohy-drate antigens such as lipopolysaccharide (LPS) O-side chainsor the alginate capsule (11, 30, 37). To our knowledge, nocomparative analysis of the vaccine efficacy of flagellin versusthat of flagella has been described for P. aeruginosa or otherpathogens. Thus, it is not clear if it is flagellum or flagellin thatis the best vaccine candidate, if either or both could be effec-tively utilized as a component of a conjugate vaccine, and if useof these vaccines could induce a state of enhanced susceptibil-ity to infection by blocking flagellin-TLR5 interactions thatpromote effective innate immunity, as was found with antibod-ies induced by a DNA vaccine encoding P. aeruginosa flagellin(33). The purpose of this study was to compare whether im-munity to P. aeruginosa flagella and that to flagellin are com-parable or distinct and to evaluate if antibodies neutralizingTLR5 activation are induced and whether this impacted TLR5activation by flagellin, flagella, or intact P. aeruginosa cells.

MATERIALS AND METHODS

Bacterial strains. The P. aeruginosa strains used for these studies were asfollows: PAK, a serogroup O6, type a flagellated strain; PA01, a serogroupO2/O5, type b flagellated strain; PAK�fliC, a fliC deletion strain of P. aeruginosastrain PAK (9); and PA01�fliC, a gentamicin insertion mutant carrying themutation in the fliC gene of P. aeruginosa strain PA01 (17); PA01ExoU�, a PA01strain expressing the ExoU cytotoxin (1); and clinical isolates CF6, -12, -19, -20,-38, and -42, obtained from CF patients who participated in a flagellar vaccineclinical trial (12) and, despite having been immunized with flagella, nonethelesswere infected with flagellum-positive strains. The flagellar mutant strains wereprovided by Reuben Ramphal (University of Florida).

Animals. C57BL/6 mice were obtained from Charles River Laboratories. NewZealand White rabbits were from Millbrook Breeding Labs, Amherst, MA. Allanimal studies were conducted in accordance with protocols approved by theHarvard Medical Area Institutional Animal Care and Use Committee.

Purification of flagella. Flagella were purified from P. aeruginosa strains aspreviously described, with some modifications (25, 40). Briefly, bacteria weregrown statically overnight in tryptic soy broth (TSB), harvested by centrifugation,and resuspended in cold phosphate-buffered saline (PBS). Flagella were re-moved from the cells by shearing in a cold Waring blender for 35 s. The cellswere separated from the flagella by centrifugation at 16,000 � g for 15 min. Thesupernatant thus obtained was then ultracentrifuged at 40,000 � g for 3 h.Flagella, obtained as a pellet, were then suspended in a minimum amount of PBSand filtered through a 0.45-�m-pore-size Millipore filter. Flagellar preparationswere also passed through a polymyxin B column to remove lipopolysaccharide

(LPS) contaminants and analyzed by a Limulus amebocyte lysate assay withEscherichia coli O113:H10 endotoxin as the standard (Cape Cod Associates) andwith P. aeruginosa LPS used as a comparator. No endotoxin was detected at alevel of �0.01% in the flagellar preparations. Flagella purified by this method,similar to that used in the vaccine trial of CF patients (12), also contain the FliDcap protein and components of the flagellar basal apparatus (4).

Purification of flagellin. Recombinant flagellins were purified from E. coliBL21(DE3) carrying the pET15BVP vector with the His-tagged type a or b fliCgene as previously described (39).

Immunization of rabbits. Female New Zealand White rabbits were immunizedsubcutaneously (s.c.) with 100 �g of flagellin or 10 �g of flagella suspended inincomplete Freund’s adjuvant (Sigma) on days 1 and 8. The rabbits were boostedintravenously with three 100- or 10-�g doses the following week. Further boosterdoses of 100 or 10 �g were given intravenously at intervals of 2 to 4 months.

ELISA. Enzyme-linked immunosorbent assay (ELISAs) were performed bystandard methods as described previously (30). In brief, microtiter plates werecoated with flagellin or flagella (0.2 M carbonate buffer, pH 9.6) and keptovernight at 4°C. Between incubation steps, plates were washed three times withPBS containing 0.05% Tween 20 (PBS-Tw). Blocking was performed with 1%bovine serum albumin (BSA) in PBS overnight at 4°C. An alkaline phosphataseconjugate goat anti-rabbit IgG antibody diluted 1:1,000 was used as a secondaryantibody, and p-nitrophenyl phosphate was used as a substrate (1 mg/ml indiethanolamine buffer, 0.5 mM MgCl2 [pH 9.8]; Sigma). After 60 min of incu-bation at 37°C, the absorbance was measured at 405 nm. ELISA titers werecalculated by linear regression analysis of the average of duplicate measure-ments; the titer was the serum dilution giving a final optical density (OD) valueof 0 at 405 nm as calculated from the linear regression curve.

Opsonophagocytic assay. An opsonophagocytic assay was used as previouslydescribed (2, 18) with the following modifications: to prepare bacteria for use inthe assay, 3 ml instead of 10 ml of TSB was inoculated with bacteria from atryptic soy agar (TSA) plate grown overnight at 37°C, with the inoculum placedin the TSB tubes, and bacteria were grown statically at 37°C until an OD at 650nm (OD650) of 0.2 was obtained; RPMI medium was used with 10% heat-inactivated fetal bovine serum and 25 mM HEPES as the diluent; and infantrabbit serum adsorbed with the target strain for 1 h at 4°C was used as acomplement source instead of human serum. The opsonic activity of immunesera was compared to that of sera obtained before vaccination. Negative controlsincluded tubes from which polymorphonuclear leukocytes, complement, or se-rum was omitted. After the 90 min of incubation, a 50-�l portion was removedand diluted in TSB containing 0.05% Tween 20 as previously described (2). Theopsonic activity of the serum was calculated as follows: [1 � (CFU immuneserum at 90 min/CFU of preimmune serum at 90 min)] � 100 (37). To determinethe amount of serum needed to kill 50% of the target bacteria (EC50), dilutionswere log transformed and logistic regression analysis used to estimate the EC50

and 95% confidence interval (CI) using the PRISM 4 software package.All studies involving human participants were approved by the Partners Health

Care Institutional Review Board, and all participants gave written consent.Motility and motility inhibition assays. Motility assays were performed as

previously described (7, 21), with some modifications. Bacteria were grown inTSB (with 100 �g gentamicin/ml for fliC mutants) statically at 37°C. Approxi-mately 105 log-phase organisms were inoculated onto plates made with lysogenybroth (LB) and 0.3% agar in the presence or absence of various dilutions ofantisera raised to flagellin or flagella and incubated at 30°C for 18 h. Results werephotographically recorded after 18 h.

Flagellar typing of CF strains. Flagellar typing of CF strains was accomplishedby PCR amplification of the central region of the flagellin gene using primersspecific for N-terminal (CW46 [5�-GGCCTGCAGATCNCCAA-3�]) and C-ter-minal (CW45 [5�-GGCAGCTGGTTNGCCTG-3�]) conserved regions as previ-ously described (8, 43). The genomic DNA was isolated by using the Wizardgenomic DNA purification kit (Promega) according to the manufacturer’s in-structions.

Mouse model of infection. (i) Passive vaccination. Female C57BL/6 mice, 6 to8 weeks old, were given 200 �l of antibodies raised to either flagellin or flagellaor given 200 �l of normal rabbit serum (NRS) i.p. 48, 24, and 4 h prior toinfection.

(ii) Active vaccination. Female C57BL/6 mice, 6 to 8 weeks old, wereimmunized intranasally (i.n.) as described previously (11). Briefly, prior tovaccination, mice were anesthetized by i.p. injection of 0.2 ml of xylazine (1.3mg/ml) and ketamine (6.7 mg/ml) in sterile water. For active vaccination,mice were given 2 �g of either flagellum type a or b, 2 �g of BSA, or 0.2 �gof LPS i.n. in a 12.5-�l volume (6.25 �l per nostril) on days 1, 8, and 15. Micewere infected on day 40 � 2.

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Survival studies. For bacterial challenge, P. aeruginosa strains were grown onTSA plates overnight, bacteria from these plates were inoculated into TSB (with400 �g of carbenicillin/ml for the PA01ExoU� strain) at an OD650 of 0.1 andgrown statically to an OD650 of 0.2 at 37°C. Bacteria were recovered by centrif-ugation, resuspended in PBS to the desired dose for infection, and washed threetimes in this buffer. Prior to administration to animals, bacterial cells were platedon MacConkey agar plates to determine the concentration. For infection, 25 �lof P. aeruginosa, prepared as described above, was slowly pipetted onto the naresof anesthetized mice, and animals were observed for survival twice a day for upto 5 days (1, 34).

Serum collection. Mouse sera were collected as described previously withsome modifications (10). Briefly, blood samples were collected from the tail veinof each mouse after warming with a heat lamp and making a small nick with asterile scalpel. Serum was separated from cells by centrifugation at 1,700 � g for10 min, and the sera were stored at �20°C until use.

Cell culture and Toll-like receptor 5 activation assay. A TLR5-expressingA549 lung epithelial cell line stably transfected with a nuclear factor kappa B(NF-B) luciferase reporter plasmid (NFB-luc; Panomics, Freemont, CA) wasused to detect cellular activation by P. aeruginosa flagellin, flagella, or intactbacterial cells. These cells are human lung adenocarcinoma cells expressing aluciferase-linked reporter regulated by multiple copies of the NF-kB responseelement. The cells were grown in Dulbecco’s modified Eagle’s medium supple-mented with 10% bovine calf serum and selected by 100 �g hygromycin B/ml inT75 flasks. After 90% of confluence was reached, A549/NFB-luc cells weretransferred to 96-well solid white plates (Costar) at a concentration of 5 � 104

cells/well. Antisera were diluted 1:50 and then added along with flagellin, flagellaor P. aeruginosa live cells to the plates, which were then incubated for 5 h. After

5 h, a Steady-Glo luciferase reagent (Promega) was added, and the resultantluminescence was read in a luminometer after 10 min of incubation at roomtemperature. Lactate dehydrogenase (LDH) release and cell viability were an-alyzed using the LDH-based in vitro toxicology assay kit (Sigma-Aldrich).

Statistical analysis. Survival data for the different mouse groups were ana-lyzed by using Kaplan-Meier survival curves and the log rank test.

RESULTS

ELISA determination of immune responses. After four rab-bits were immunized with type a or b flagella or flagellin,antibody titers to those proteins were evaluated by ELISA. Allfour proteins were highly immunogenic (Table 1) and showedstrong cross-reactivity between homologous flagella and flagel-lin proteins. The antisera also cross-reacted with the heterol-ogous proteins but to a lower level than with the homologousproteins. Antibody to type a flagella or flagellin reacted betterwith type b proteins than did antisera raised to the type bproteins with the type a flagella or flagellin.

Motility inhibition assays. To determine the functional ac-tivity of the antisera to P. aeruginosa flagellin or flagella, weevaluated the ability of the antisera to inhibit motility of P.aeruginosa strains PAK and PA01. In these assays, NRS wasused as a negative control. Antisera to flagellin at a dilution of1:75 or less inhibited the motility of the corresponding type ofP. aeruginosa. Antisera to flagella had a higher titer of motility-inhibiting antibodies, showing full inhibition of motility at adilution of 1:200. The antiserum to flagellum type a showedsome cross-reactivity to flagellum type b since it inhibited themotility of strain PA01 (Fig. 1), consistent with the titer deter-minations (Table 1). The six clinical isolates from CF patientswere also tested in this assay, and the flagellar type identifiedby PCR was confirmed by showing inhibition of motility by thecorresponding antiserum at a dilution of 1:50 (data not shown).

TABLE 1. Titers of antibodies to type a or b flagella or flagellin inrabbit serum raised to either type a or type b flagella or flagellin

Serum raised to:

Titer of antibody against target antigen

Flagellumtype a

Flagellintype a

Flagellumtype b

Flagellintype b

Flagellum type a 2,172,500 920,000 139,000 167,750Flagellin type a 1,015,800 1,900,950 78,500 38,150Flagellum type b 36,520 57,000 230,000 741,200Flagellin type b 36,100 107,820 1,042,180 659,550

FIG. 1. Assessment of inhibition of motility of P. aeruginosa strains PAK and PA01 with antibodies raised to either flagellin or flagella. The wellscontaining motility agar were inoculated with the indicated P. aeruginosa strain: PA01 (type b flagella) or PAK (type a flagella) or thecorresponding strain lacking the fliC gene. Specific antisera were also added to the wells at the indicated dilution.

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Opsonic killing activity of antisera to flagellin or flagellaagainst P. aeruginosa. In an opsonophagocytic killing assayusing antibodies raised to either flagellin or flagella, we deter-mined the overall activity and estimated the serum dilutionmediating killing of 50% of the bacterial cells (EC50) (Table 2and Fig. 2). Antisera to flagellin had little to no opsonic killingactivity, with the serum raised to type a flagellin having lowopsonic killing activity (EC50 � 10) using homologous strainPAK and no killing of strain PAO1 (Fig. 2A), while an anti-

serum to type b flagellin had no killing activity (EC50 � 4)against homologous strain PA01 or heterologous strain PAK(Fig. 2B). Antiserum to polymeric type a flagella was quiteactive, with an EC50 of 937 against homologous strain PAK(Fig. 2C), while antisera to type b flagella had a modest EC50

of 19 against homologous strain PA01 (Fig. 2D). After flagellartyping by PCR of P. aeruginosa CF isolates and ascertainmentof functionality in motility assays (not shown), we randomlychose three strains of each type (for type a, CF 6, 38, and 42;for type b, CF 12, 19, and 20) to evaluate the opsonic activitiesof the antibodies raised to flagella. Antisera to type a flagellawere highly active in mediating opsonic killing of the three typea CF clinical isolates, although the estimated EC50s were lowerthan those for activity against the homologous PAK strain(Table 2). Antisera to type a flagella also had modest killingactivity against the three type b clinical isolates, comparable tothat against the type b strain PAO1. The antiserum to type bflagella did not mediate killing of the type a clinical isolates(EC50 � 4) but had modest to high activity against the flagellartype b clinical isolates (Table 2).

Survival studies. (i) Passive vaccination. Rabbit antibodyraised to either type a or type b flagellin or flagella was usedto passively immunize C57BL/6 mice i.p. After serum injec-tions, mice were challenged i.n. with a P. aeruginosa flagellartype a or b strain at approximately two times the 50% lethaldose (LD50). Antibodies raised to either type of monomericflagellin showed no protection against strain PAK orPA01ExoU� (Fig. 3A and B) or against the clinical isolateCF6 (data not shown). In contrast, antibody to polymeric

TABLE 2. Estimated dilution of serum needed to mediate killingof 50% (EC50) of the target bacterial strain in an

opsonic killing assay

Target strain

EC50 (95% CI) of serum raised to:

Type a Type b

Flagella Flagellin Flagella Flagellin

PAK (type a) 937 (215–4,086) 10 (5–19) 4 (0.7–18) �4a

CF6 (type a) 55 (29–104) �4a

CF38 (type a) 207 (10–4,139) �4a

CF42 (type a) 134 (58–312) �4a

PA01 (type b) 45 (3–7) �4a 19 (12–28) �4a

CF12 (type b) 8 (3–19) 12 (10–14)CF19 (type b) 15 (12–18) 60 (35–103)CF20 (type b) 8.9 (4–20) �256 (95% CI not

determinedfrom data)

a Opsonic killing of �30% in the 1:4 serum dilution was considered to bewithin the range of the controls lacking an essential component needed foropsonization and/or killing, and thus, titers are reported as less than 4. Blanksindicate the test was not performed because no killing (EC50 � 4) was detectedin the homologous bacterium-antibody mixture.

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 representmeans of duplicate-quadruplicate determinations, and error bars represent SEM. Based on nonlinear regression determination of the titer ofantibody needed to mediate killing of 50% of the target bacteria, comparisons showed results to be significantly (P � 0.05) different.

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type a flagella achieved 87.5% survival following challengewith strain PAK (P � 0.0001 versus results for the NRSgroup) and 43.75% survival using strain CF6 (P � 0.0028versus results for the NRS group). Antibody to the heterol-ogous type b flagella did not protect against the type astrains (Fig. 3C and E), indicating specificity of this anti-serum for the type b flagellum immunogen prepared from P.aeruginosa cells. Immunization with antibody to polymerictype b flagella resulted in 70.8% survival of strainPA01ExoU� (P � 0.0001 versus results for the NRS group)and 12.5% survival of strain CF20 (P � 0.025 versus resultsfor the NRS group). The antiserum to the type a flagellaagain exhibited some cross-reactivity with type b strains,providing 50% survival of mice challenged with strain

PA01ExoU� (P � 0.0005 versus results for the NRS group)and also resulted in an increase in the mean time to death to36 h in of mice vaccinated with the anti-flagellum type aserum and infected with type b strain CF20. This increasewas approximately 1.5 (95% CI, 1.12 to 1.88) times longerthan that for mice vaccinated with NRS (P � 0.009) (Fig. 3Dand F). However, antiserum to type a flagella did not im-prove the overall survival of mice infected with the type bclinical isolate CF20, again indicating that there was a highdegree of specificity of the antibody for engendering survivalagainst strains expressing the type a flagella isolated from P.aeruginosa cells.

(ii) Active vaccination. Since passive immunization with an-tisera to flagellin was poorly opsonic and not protective against

FIG. 3. Survival of mice passively immunized with antibody to monomeric flagellin or polymeric flagella. (A and B) Mice immunized withantibodies to monomeric flagellin and challenged with P. aeruginosa type a strain PAK ( 4.75 � 107 CFU/mouse; log rank test, anti-flagellin typea 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 versusNRS, P � 0.35; median survival, 48 h, both groups). (C to F) Mice passively immunized with antibody to polymeric flagella and challenged withP. 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 flagellaversus 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 flagellaversus 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, antibodyto type b flagella, 30 h; antibody to type a flagella, 36 h; NRS, 24) (F).

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pneumonia, we focused the active vaccination studies on theprotective activity of polymeric flagella. Mice were vaccinatedi.n. with homologous or heterologous type a or type b flagella,BSA, or homologous LPS, which was used at a dose 1/10 thatof the flagella to control for possible effects from potentiallycontaminating, albeit undetectable, LPS in the vaccines. Aftervaccination, mice were challenged i.n. 40 to 42 days later withstrain PAK or PA01ExoU� (Fig. 4A and B). Before infection,blood samples were collected from each mouse to evaluate theantibody response to the flagellar antigens. Both flagella werehighly immunogenic by the i.n. route, as determined by findinghigh serum IgG antibody titers in an ELISA (data not shown).After infection with strain PAK, 83.3% of the mice immunizedwith homologous type a flagella survived, compared with 25%of the mice in the group immunized with BSA (P � 0.0016)and 8% survival in mice immunized with a low dose of LPSfrom strain PAK (P � 0.0001). There were no survivors in thegroup of mice immunized with heterologous type b flagella(P � 0.0001). Immunization with type b flagella was highlyprotective against infection with the homologous type b flagel-lum strain PA01ExoU�, with a survival rate of 83.3%, whereasin the group of mice immunized with BSA only, 8% of micesurvived (P � 0.0001) and there were no survivors in miceimmunized with LPS from strain PA01 or type a flagella (P �

0.0001, both comparisons). Immunization with type a flagellaalso protected against infection with type a strain CF6, with asurvival rate of 41.67%, compared to 8.33% for the groupimmunized with BSA (P � 0.030), 16.67% for immunizationwith LPS (P � 0.09), and no survivors in the group of miceimmunized with type b flagella (P � 0.0007) (Fig. 4C). Immu-nization with type b flagella showed borderline protectionagainst type b strain CF20, with a survival rate of 25%, while inthe group of mice immunized with BSA, only 8.33% of micesurvived (P � 0.09) and there were no survivors among themice immunized with homologous LPS or type a flagella (P �0.013) (Fig. 4D). These results indicate high-level, flagellar-type-specific protection against pneumonia and death follow-ing i.n infection with strains PAK and PA01ExoU� but clearlyless protection when the challenge was with clinical isolatesobtained from CF patients that had been immunized with aflagellar vaccine but nonetheless became colonized with P.aeruginosa. Since neither LPS nor immunization with the het-erologous flagella provided protection, the protective immuneresponses of the mice were clearly specific to the immunizingantigen and not due to potential cross-reactive antibodies elic-ited by contaminating antigens.

(iii) TLR5 activation. We next evaluated whether antibodyto flagella and/or flagellin inhibited TLR5 activation. In

FIG. 4. Survival of mice after active vaccination with flagella. (A) Mice challenged with P. aeruginosa PAK ( 2.2 � 107 CFU/mouse) (log ranktest, 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 aflagella, 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 aflagella, BSA, and LPS, 36 h). BSA- and LPS-immunized mice served as the controls in all experiments. Results are represented in Kaplan-Meiersurvival curves and analyzed by the log rank test. “n” refers to the number of animals.

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these assays, purified flagellin, as expected, was a morepotent activator of TLR5 than flagella (Fig. 5), which arewell known to release flagellin monomers during storage (6),making it difficult to determine if the activation was due tointact flagella or released flagellin. When comparing activa-tion mediated by type a versus type b flagellin, there were nodifferences in the degree of luminescence, but polymerictype a flagella activated the cells to a higher level thanpolymeric type b flagella, particularly at concentrations

lower than 1 �g/ml (Fig. 5). This comparative difference hasbeen previously noted (42). When antibodies to flagellin orflagella were added to test for inhibition of TLR5 activationby flagellin or flagella, antibody to monomeric flagellin hada higher titer with greater neutralization of TLR5 activationthan did antibodies raised to polymeric flagella (Fig. 6). Inthese assays, we noted the NRS control had some ability toactivate the cells, which could be due to serum factors as hasbeen described for TLR4 activation by LPS and CD14 andthe LPS binding protein (LBP) (16, 38).

We also assessed the activation of TLR5 by live P. aeruginosacells, the obvious entity which would trigger innate immunityduring infection, and the inhibition of this activation by thecorresponding antisera. In these assays, P. aeruginosa PAK andPA01 bacteria activated the NF-B activity of the cells (Fig.7A); however, strain PAK was a better activator than strainPA01, which coincided with the results seen when testing thepurified flagellar proteins from these strains. Analysis of LDHrelease as an indicator of cytotoxicity during incubation withthe bacterial cells showed there was no more than a 10% differ-ence in LDH release between cells incubated with bacteria andcontrols incubated without bacteria (data not shown). The �fliCmutant bacterial strains used as controls did not promote anyNF-B-dependent activation, which also confirmed the specificityof the cells for responding to flagellin (Fig. 7A). Inhibition of theactivation of TLR5 by antibody to flagella or flagellin showed thatthe two antisera had comparable activities at inhibiting TLR5

FIG. 5. Activation of TLR5 by P. aeruginosa flagellin or flagella.Addition of the indicated amount of purified protein induced theproduction of luciferase initiated by TLR5-dependent activation ofNF-B and binding of nuclear translocated activated NF-B fragmentsto cognate sites in the luc gene promoter. The points are the means ofquadruplicate 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 ofTLR5 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 antibodyto 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 activationof 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.

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activation by strain PAK cells (Fig. 7B). Notably, we did not findantibody to either flagella or flagellin capable of inhibiting theactivation of TLR5 by P. aeruginosa strain PA01 cells (Fig. 7C)compared to the activity of added NRS, which had a small inhib-itory effect on its own. When we tested antiserum to type bflagellin or flagella along with two other type b strains in theTLR5 activation assay, we obtained an identical result (data notshown). Thus, while antibody to both type a flagella or flagellininhibited TLR5 activation by strain PAK, no such inhibitor effectwas seen with strain PA01 or other type b flagellum strains of P.aeruginosa.

DISCUSSION

The goals of this study were to compare the immunogenicityand functionality of flagellin and flagella as possible candidates

for a vaccine against P. aeruginosa pneumonia and also todetermine if such immunogens might give rise to antibodiesthat could interfere with the host’s ability to detect the pres-ence of flagellated pathogens via TLR5 activation (15, 32, 35).Prior work with animals (3, 13, 27) and CF patients lackingdetectable P. aeruginosa colonization (12) has validated thevaccine potential of flagella. Vaccines based on monomericflagellin have shown some efficacy in animals when a DNAvaccine encoding P. aeruginosa flagellin lacking TLR5 agonistactivity is used (33), as has a multimeric construct ofOprF(311-341)-OprI-flagellin fusion proteins (42) in models ofburn wound and pulmonary infection. However, no direct com-parison of the protective efficacy against infection by immuni-zation with flagellin or flagella has been reported for P. aerugi-nosa or, as far as we can determine, for other microorganisms.In vitro studies suggest that the protective activity of antibodyto flagella or flagellin correlates with inhibition of motility (24,27, 28) and opsonic killing (42). Here we found that polymericflagella, which by nature contain, in addition to the polymer-ized FliC protein, components such as the FliD cap proteinand possible basal body components, were clearly superior tomonomeric flagellin at inducing antibodies that inhibited mo-tility, mediated opsonic killing, and provided protective immu-nity against acute lung infection. Also, the specificity of theprotection for strains expressing only the homologous flagellartype indicated that potentially contaminating antigens inflagellar preparations isolated from P. aeruginosa cells were notcontributing to the protective effects observed. Additionally,immunization with flagella induced lower titers of antibodythat could interfere with the TLR5 agonist activity of flagellinand flagella, but only antibody to type a flagella and flagellininhibited TLR5 activation by whole bacterial cells. These find-ings suggest a superiority of intact flagella over monomericflagellin as a component of a P. aeruginosa vaccine.

In evaluating effects on P. aeruginosa motility and opsonickilling in vitro, we found flagellar-type-specific activity withantibodies to type b antigens, whereas the antibodies raised totype a flagella had some activity against type b strains. Sinceamino acid segments of the two flagella types are partiallysimilar, it is possible that the type a flagellum vaccine wasbetter able to induce antibodies to these shared components.Another potential basis for these differences may be glycosyl-ation of flagellar proteins (5), wherein the type a flagellum isglycosylated by larger and more heterogeneous oligosaccha-rides than type b (40), possibly enhancing its immunogenicity.In addition, the lack of the glycan groups on the recombinantmonomeric flagellins could also partly explain the poor abilityof the antibodies raised to these proteins to promote opsonickilling. Nonetheless, for both motility inhibition and opsonickilling, the intact flagella were superior to monomeric flagellinat inducing antibodies mediating these in vitro correlates ofprotection.

In the mouse pneumonia model, we showed that passiveimmunization with antibodies to flagellin did not confer pro-tection against infection with either type of P. aeruginosa. Thisoutcome is different from that in the work of Saha et al. (33),who reported that immunization with a flagellin DNA vaccineprotected against heterologous but not homologous bacterialchallenge. This is curious in that almost all prior studiesshowed protection following flagellar vaccination was type spe-

FIG. 7. Effects of antibody to flagella or flagellin on activation ofTLR5 by whole bacterial cells. (A) Luciferase production due to acti-vation of TLR5 by P. aeruginosa strain PAK, PA01, PAK�fliC, orPA01�fliC. Both wild-type strains induced TLR5 activity, whereas thestrains lacking flagella were devoid of agonist activity. (B) Activation ofTLR5 by P. aeruginosa strain PAK bacteria and inhibition by antiseraraised to either type a flagella or flagellin at a 1:50 dilution. (C) Ac-tivation of TLR5 by P. aeruginosa strain PA01 bacteria and inhibitionby antibody to type b flagella or flagellin at a 1:50 dilution. NRS wasused as a control. The points are means of triplicate determinations,and error bars represent SEM.

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cific (3, 13, 19, 27). The DNA vaccine construct may be supe-rior at stimulating cross-protective humoral and/or cellular im-mune responses compared to intact flagellin protein, or activeimmunization with a DNA vaccine may provide results supe-rior to those of passive immunization. Weimer et al. (42)obtained enhanced clearance of P. aeruginosa strain PA01 fol-lowing immunization of mice with a construct of OprF311-341-OprI-flagellins, although the challenge dose was insufficient tocause mortality in the controls immunized with OprF311-341-OprI vaccine lacking flagellins. Similarly, immunization ofyoung African green monkeys with this construct elicited se-rum antibodies that when passively administered to mice couldpromote clearance of P. aeruginosa from the lungs (41). How-ever, since our goal was to compare flagellin-mediated immu-nity to that induced by polymeric flagella, we were able tovalidate a superior effect of the latter in passive transfer studieswherein injection of rabbit antibodies raised to flagella dem-onstrated high levels of protection in mice against lethal lunginfection.

Active vaccination with intact flagella also showed high lev-els of type-specific, LPS-independent protection in a mousepneumonia model and modest but less protection against mo-tile clinical isolates from CF patients. These isolates were ob-tained from CF patients enrolled in the flagellar vaccine trial(12) who, in spite of vaccination, nonetheless became colo-nized with P. aeruginosa. We suspected these strains might beless susceptible to protection mediated by flagella derived fromstrains PAK and PA01, and the findings in our mouse studiesbear this out. This could have important consequences forfuture vaccine trials incorporating flagella as vaccines and sug-gests that there may be additional flagellar components neededin a comprehensive vaccine, such as those expressing differentsubtype antigens on type a flagella (3). Since virtually all priorstudies in this area have only evaluated protection againststrain PAK or PA01, conclusions about the utility of flagellinor flagella as a vaccine have to be tempered with the lack of acomprehensive demonstration of efficacy against multiplestrains, including clinical isolates, and, now that they are avail-able, clinical isolates from flagellum-vaccinated CF patientsunable to resist colonization by P. aeruginosa.

Another concern related to use of flagellin or flagella asvaccines is whether they will induce antibodies that interferewith TLR5-mediated innate immunity (15, 22, 32, 35, 36),which has been suggested by the studies of Saha et al. (33).However, the experimental design of that study is not infor-mative as to the potential for antibody to flagella or flagellin tointerfere with innate immunity during an actual infection withlive P. aeruginosa cells. These investigators used purified, re-combinant flagellin to enhance TLR5-mediated innate immu-nity by first incubating it with either antibody to wild-typeflagellin or antibody raised to the R90A flagellin variant lack-ing the TLR5 binding domain. They subsequently inoculatedBALB/c mice with this flagellin-antibody mixture 2 h prior tochallenge with live P. aeruginosa PA01. The flagellin mixedwith antibody to wild-type flagellin was less able to conferprotection from lung infection than was flagellin mixed withantibody to the R90A variant, presumably due to inhibition ofTLR5-mediated innate immune responses. However, this ex-periment did not indicate if antibody to flagellin inhibited

TLR5 activation from whole bacteria during infection, thusincreasing the animal’s susceptibility to infection.

When testing the activation of TLR5 by P. aeruginosa bac-terial cells, strain PAK was a more potent activator of thereceptor than was strain PA01, consistent with the findingsusing purified flagella from these strains. However, for unclearreasons, while antibody to both flagella and flagellin couldinhibit in a dose-dependent manner TLR5 activation mediatedby strain PAK, neither of them could inhibit activation medi-ated by PA01 or by two other type b flagellum strains. Sincethere was no luciferase signal from cells infected with the �fliCstrains, other P. aeruginosa PAMPs, such as LPS, were notactive in this assay. The mechanisms that might explain theinability of antibody to flagella or flagellin to inhibit the acti-vation of TLR5 by type b strains is not clear, but it may notnecessarily be related to preventing TLR5 binding but ratherperhaps to some other property of antibody to flagella orflagellin, such as inhibition of motility. In this regard, type astrains such as PAK may be less able to interact with the cellsif motility is inhibited by the antibody to flagella, whereas thetype b strains may either be less inhibited in their overallmotility by antibody or use an alternative means of locomotion,such as pilus-mediated twitching motility, to interact with cells,or the antiserum to type b flagella or flagellin may be lesspotent at inhibiting motility than antibody to type a flagella,although this was not apparent in our in vitro motility inhibitionassays.

Taken together, it seems that flagella, composed primarily ofthe FliC protein but also containing the type-specific FliD capprotein and basal body components, would be a better candi-date for a vaccine against P. aeruginosa than flagellin, sinceimmunization with flagella was demonstrated to be more pro-ficient in generating flagellar-type-specific antibodies that in-hibit motility, mediate opsonic killing, and protect againstacute P. aeruginosa lung infection. In addition, although theantibodies to flagella inhibited activation of TLR5, they wereless potent than antibodies to flagellin in this regard, minimiz-ing the potential for interference with the induction of innateimmunity mediated by activation of TLR5. Of note, however,use of flagella from strains PAK and PA01 as vaccines pro-vided only modest protection against clinical isolates fromflagellar-vaccine-immunized CF patients who nonetheless be-came colonized with flagellated P. aeruginosa. This suggestsadditional flagellar antigens may need to be incorporated intoa comprehensive vaccine or strains other than the prototypePAK and PA01 may need to be utilized as a source for theflagellar antigens to find some that either are more immuno-genic or give rise to more cross-reactive antibodies. Identifyingthe optimal formulation of the components of flagella neededfor a maximally effective P. aeruginosa vaccine should enhancethe utility of this approach for future evaluations.

ACKNOWLEDGMENTS

This work was supported by NIH grants AI048917 and HL058398.

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Editor: J. N. Weiser

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