The Journal of Immunology

Blocking of the TLR5 Activation Domain Hampers Protective
Potential of Flagellin DNA Vaccine1
Sukumar Saha,2* Fumihiko Takeshita,2,3* Tomoko Matsuda,* Nao Jounai,* Kouji Kobiyama,*
Tetsuya Matsumoto,† Shin Sasaki,* Atsushi Yoshida,* Ke-Qin Xin,* Dennis M. Klinman,‡Satoshi Uematsu,§ Ken J. Ishii,¶ Shizuo Akira,§ and Kenji Okuda*
Flagellin is a key component of the flagella of many pathogens, including Pseudomonas aeruginosa. Flagellin is an attractive
vaccine candidate because it is readily produced and manipulated as a recombinant protein and has intrinsic adjuvant
activity mediated through TLR5. Although DNA vaccines encoding native Pseudomonas B-type (FliC) or A-type (FlaA)
flagellin are strongly immunogenic, the resultant Ab response interferes with the interaction of homologous flagellin with
TLR5. This reduces the ability of the host to clear homologous, but not heterologous, flagellin-expressing P. aeruginosa. To
circumvent this problem, a DNA vaccine encoding a mutant FliC R90A flagellin was developed. The mutant Ag encoded bythis vaccine was highly immunogenic, but its ability to interact with TLR5 was reduced by >100-fold. Vaccination with thisflagellin mutant DNA vaccine induced cross-reactive Abs against both FliC and FlaA, but few Abs capable of interfering withTLR5 activation. The flagellin mutant DNA vaccine provided excellent protection against both FliC- and FlaA-expressing P.
aeruginosa. These findings suggest that vaccines against flagellated pathogens should avoid inducing Abs against TLR5 andraise the possibility that flagellated bacteria evade host elimination by facilitating the production of Abs that reduce thehost’s ability to mount an innate immune response. The Journal of Immunology, 2007, 179: 1147–1154.

F

lagella are conserved organelles that play a critical role inproviding motility to diverse bacterial species, including
Pseudomonas aeruginosa (1). Reducing flagellar activity
restricts motility, and thus limits an organism’s ability to invade
and effectively colonize a new host (2). Flagella are effectively
targeted by the host immune system, and bacteria that delete or
mutate their flagella can cause severe persistent infection (as in thecase of Legionella pneumonia and Salmonella typhimurium) (3, 4).
Flagellin, a key protein component of flagella, can also trigger
an innate immune response by interacting with a cell surface receptor TLR5 (5). The domains of flagellin involved in interactingwith TLR5 have been investigated. The NH2- and COOH-terminal
regions of flagellin are well conserved among various Gram-neg-*Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, Japan; †Departmentof Microbiology, Tokyo Medical University School of Medicine, Tokyo, Japan; ‡Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Foodand Drug Administration, Bethesda, MD 20892; §Department of Host Defense, Research Institute for Microbial Diseases, Osaka, Japan; ¶Department of MolecularProtozoology, Research Institute for Microbial Diseases, Osaka, Japan; and Exploratory Research for Advanced Technology, Akira Innate Immunity Project, Japan Science and Technology Agency, Osaka, Japan
Received for publication March 26, 2007. Accepted for publication May 5, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a Grant-In-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and Technology and the Yokohama
Medical Foundation (Yokohama City University Center of Excellence Program to
N.J., 18590432 to F.T., and 18659126 to K.O.), the National Institute of Biomedical
Innovation (to K.O.), Strategic Research Project of Yokohama City University(K18022 to F.T.), Advancement of Medical Sciences from Yokohama Medical Foundation (to F.T.), and Japan Science and Technology Agency (131-1 to F.T.).
2

S.S. and F.T. contributed equally to this work.

ative bacteria (6). In studies of S. typhimurium FliC and Salmonella muenchen flagellin, deletion of either the NH2- or COOHterminal regions abolishes the interaction with TLR5. A singleamino acid substitution in either region results in a loss of proinflammatory activity, suggesting that the structure at both ends ofthe molecule contribute to TLR5 interactions (7, 8). Characterization of flagellin chimeras containing domains from “active” (Salmonella) and “inactive” (Helicobacter) types demonstrated that
type-specific NH2- and COOH-terminal domains (particularly aa
89 –96 at the NH2 terminus) play a critical role in TLR5 activation.
Similarly, aa 83–97 in the NH2 terminus of P. aeruginosa A-typeflagellin (FlaA)4 was essential for TLR5 activation (9). Taken together, these findings suggest that the tertial structure generated bydomains in both the NH2 and COOH terminus regions are criticalfor TLR5 activation by flagellin.
Activating TLR5 expressed by epithelial cells, macrophages, or
dendritic cells initiates a signaling cascade mediated through an
adaptor molecule, MyD88, IL-1R-associated kinase, TNFR-associated factor 6, and I␬B kinase. This process culminates in thestimulation of innate immune responses characterized by the production of proinflammatory cytokines including TNF-␣, IL-6, IL10, and IL-12 and cell surface molecules including MHC, CD40,
B7.1, and B7.2. Activation of this cascade has been shown to
improve the induction of vaccine-specific adaptive immune
responses (10 –20).
Based on these findings, it was postulated that flagellin could bea very effective vaccine candidate. Flagellin Ags should trigger anadaptive immune response that targets multiple pathogens, while
the NH2- and COOH-terminal domains of the protein should act as
intrinsic adjuvants, stimulating a TLR5-dependent innate immune
response. Although there have been reports of full-length flagellin
being used to induce the production of anti-flagellar Abs (21, 22),3

Address correspondence and reprint requests to Dr. Fumihiko Takeshita, Department of Molecular Biodefense Research, Yokohama City University Graduate Schoolof Medicine, Yokohama 236-0004, Japan. E-mail address: takesita@yokohama-cu.
ac.jp
www.jimmunol.org

4
Abbreviations used in this paper: FlaA, A-type flagellin; WT, wild type; FliC, Btype flagellin; p.i., postinfection; MPO, myeloperoxidase.

1148

FLAGELLIN IMMUNIZATION BLOCKS INNATE IMMUNE RESPONSES

there are no reports of full-length TLR5-activating flagellin protecting against subsequent bacterial infection. Indeed, vaccination
studies indicate that flagellin-deficient mutant Salmonella induce
superior protection when compared with wild-type (WT) parental
bacteria (23, 24). Yet, when anti-flagellar Abs were raised by immunization with truncated forms of P. aeruginosa flagellin or acomponent vaccine targeting Campylobacter jejuni flagellin, protection against lethal doses of P. aeruginosa and C. jejuni wasagain observed (25, 26). These findings, and preliminary studies in
our laboratory, suggested that full-length flagellin protein capableof interacting with TLR5 was limited in its ability to induce a
protective immune response.
To clarify the mechanisms underlying the protective potential offlagellin, a series of DNA vaccines encoding FlaA and B-typeflagellins (FliC) from different serotypes of P. aeruginosa (PAKand PAO1) (27) were generated and characterized in a murine
model of acute pneumonia. Results show that these flagellin vaccines efficiently protected mice against heterologous, but not homologous, bacterial challenge. Each flagellin vaccine raised Abs
that cross-reacted with both types of flagellin, but also Abs thatprevented TLR5 activation in a type-specific manner (thereby
hampering innate bactericidal activity). A mutagenized form offlagellin (FliC R90A) that retained the immunogenicity of the parent molecule, but had significantly reduced ability to interact with
TLR5, conferred optimal immune responses against infection byboth strains of P. aeruginosa.

Materials and Methods
Bacterial strains and culture conditions
P. aeruginosa strains PAO1 and PAK were used for this study. PAO1 andPAK express different virulence factors, such as pili, flagella, and typeIII-secreted exoenzymes. All bacterial strains were cultured in Terrific
Broth (Sigma-Aldrich) at 37°C for 16 h. For the intranasal challenge, thebacterial culture was centrifuged at 3000 ⫻ g for 10 min, and the pellet waswashed twice in PBS and resuspended in PBS at an OD of 1.0 at 550 nm.
Thirty or 40 ␮l of this suspension corresponded to ⬃2 LD50 doses for both
PAO1 and PAK.

Construction of mammalian expression plasmids for vaccination
Genomic DNA of the P. aeruginosa strain PAO1 and PAK were prepared
as described previously (28). The fliC of PAO1 and flaA of PAK were PCR
amplified and introduced into the mammalian expression plasmid pFLAGCMV5 (Sigma-Aldrich) to obtain fliC and flaA genes fused to the FLAGsignal sequence. The DNA fragments of fliC-FLAG and flaA-FLAG werethen introduced into the EcoRI site of the pGACAG vector (29) to obtain
pGACAG-FliC and pGACAG-FlaA. All of the plasmids were transformed
into Escherichia coli DH5␣ and purified using the Endo-free Maxiprep kit(Qiagen).

Generation of flagellin mutants
Several single alanine point mutations both at the NH2 (L88A, R90A, andQ97A) and COOH (V404A, R415A, Q422A, N423A, and F425A) terminus were introduced by a standard PCR-mutagenesis strategy (30). All
mutations were verified by DNA sequencing. Recombinant proteins wereexpressed in E. coli DH5␣ and purified as described elsewhere (31). Contaminated endotoxins were removed using Detoxi-Gel (Pierce). Finally, theamount of contaminated endotoxin was ⬍0.003 ng/mg protein.

Immunization and challenge schedule
⫹/⫺

⫺/⫺

Eight-week-old female BALB/c, C57BL/6 TLR5 , or TLR5
mice (5)were used for this study. After being anesthetized with a ketamine andxylazine mixture, the mice (eight mice per group) were immunized with theplasmid solution (1 ␮g/␮l saline, 50 ␮l/muscle) containing 50 ␮g
pGACAG (control empty plasmid), pGACAG-FliC, pGACAG-FliCR90A, orpGACAG-FlaA by an i.m. electroporation method as described previously(32). A booster immunization was given at 4 wk after the first immunization. Two weeks after the final immunization, the mice were anesthetizedand challenged intranasally with 2 LD50 of PAO1 or PAK. The mortalityof the challenged mice was monitored for the subsequent 10 days. All of
the animal experiments were approved by the institutional animal care andwelfare committee, and the mice were treated according to the institutional
animal care guidelines.

ImmunoblottingSpecificity of serum IgG was analyzed by using immunoblots of flagellaprepared from PAO1 and PAK as described previously (29, 33). After
separation by SDS-PAGE, samples were blotted on polyvinylidene difluoride membranes. The membranes were blocked with 10% skim milk andthen reacted with mouse sera from various groups of vaccinated mice.
After incubation with HRP-labeled anti-mouse IgG Ab, the blots werereacted with ECL plus substrate (Amersham Biosciences) and then analyzed by autoradiography.

Purification of flagella
Flagella was isolated and purified from the PAO1 and PAK strains of P.
aeruginosa according to the method described previously (34), with somemodifications. P. aeruginosa PAO1 or PAK was cultured at 37°C for 14 hand centrifuged at 10,000 ⫻ g for 15 min. Over a period of 2 h, ammonium
sulfate was added to the supernatant to reach 40% saturation. After centrifugation at 20,000 ⫻ g for 40 min, the pellets were resuspended in 8 ml
of 50 mM Tris-HCl (pH 9.5) containing 0.5 mM DTT and then dialyzed
extensively against the same buffer. After centrifugation at 27,000 ⫻ g for
40 min, the pellet (flagella) was resuspended in PBS, and stored at ⫺80°C
until use.

ELISA
Flagella-specific Ab was determined by ELISA as described previously(32). Flat-bottom 96-well plates (Nunc) were coated with flagella diluted to
2 ␮g/ml in bicarbonate buffer (pH 9.6; FliC or FlaA) at 4°C for 18 h. Plates
were then blocked with PBS containing 0.05% Tween 20 and 0.5% BSA
for 2 h and incubated with appropriately diluted serum 37°C for 2 h. Plates
were washed three times with PBST and incubated with HRP-labeled antimouse IgG at 37°C for 2 h. After washing, substrate buffer containing urea
peroxidase and tetramethylbenzidine (Sigma-Aldrich) was added and incubated at 25°C for 15 min. The plates were read using a spectrophotometer (Benchmark Plus; Bio-Rad) at an OD of 405 nm, and the titer was
generated using a high-standard antisera. TNF-␣ levels in the bronchoalveolar lavage fluid were measured according to the manufacturer’s protocol (eBioscience).

Quantification of P. aeruginosa in the lungTwo weeks after final immunization, the mice were anesthetized and inoculated intranasally with 5 ⫻ 105 CFU of PAO1 or PAK in 30 ␮l of PBS.
Twenty-four hours postinfection (p.i.), the mice were sacrificed under a
CO2-rich atmosphere, and then the lungs were removed and homogenizedin 4 ml of PBS under sterile conditions. Serial 10-fold dilutions of lunghomogenates were plated on Luria-Bertani broth agar plates (SigmaAldrich), incubated at 37°C for 24 h, and then the number of colonies wasenumerated (29).

Myeloperoxidase (MPO) assay
The MPO assay was used to determine the levels of neutrophil infiltration
into the lung as described previously (35). In brief, the lungs were homogenized in 50 mM HEPES buffer (4 ␮l/mg lung) and centrifuged at
20,000 ⫻ g at 4°C for 30 min. The pellet was suspended and homogenizedin 0.5% cetyltrimethylammonium chloride (4 ␮l/mg lung tissue). The preclear extract was collected and samples in duplicate (75 ␮l) were mixed
with equal volumes of the substrate buffer containing 3 mM 3,3⬘,5,5⬘tetramethylbenzidine dihydrochloride, 120 ␮M resorcinol, and 2.2 mM
H2O2 for 2 min. The reaction was stopped by adding 150 ␮l of 2 M H2SO4
and the OD of 450 nm was measured.

Histopathology of lungs
After being intratracheally inoculated with 4% paraformaldehyde, thelungs were removed and fixed in 4% paraformaldehyde for 24 h. Frozen
sections (thickness, 15 ␮m) were subjected to H&E staining and then analyzed under microscopy.

Luciferase assay
Luciferase assay was performed according to a previously describedmethod (36). In brief, HEK293 cells grown in 96-well plates were transiently transfected with 40 ng of mouse or human TLR5 expression plasmid in the presence of 5 ng of pNF-␬B-Luc (Stratagene) and pTK-RL

The Journal of Immunology

1149

FIGURE 1. Characterization of flagellin mutants. A, Amino acid sequence of P. aeruginosa FliC. The sequences shaded with red indicate the regions
from ␣ helix whose intramolecular conformation corresponds to the TLR5 activation domain in S. typhimurium FliC. B and C, HEK293 cells weretransiently transfected with either mouse or human TLR5 expression plasmid plus NF-␬B-dependent luciferase expression plasmid. Cells were thenstimulated with FliC WT or mutants (L88A, R90A, Q97A, V404A, or F425A) at the concentration of 10 –500 ng/ml, and luciferase activity was measured.
The graph shows the mean ⫾ SD; ⴱ, p ⬍ 0.01 by Student’s t test. D, Mice were intranasally inoculated with PBS alone (control) or 1 ␮g of the recombinant
protein of FliC WT or mutants (L88A, R90A, Q97A, V404A, or F425A). Four hours after inoculation, the bronchoalveolar lavage fluid was collected andTNF-␣ concentration was measured by ELISA. Data show the mean ⫾ SE, n ⫽ 6 mice/group, ⴱ, p ⬍ 0.05 by Student’s t test. E, Reactivity of anti-FliC
WT IgG raised in BALB/c mice with the recombinant protein of FliC WT, L88A, R90A, or FlaA was examined by ELISA. Two or three independent
experiments gave similar results.

(Promega) using FuGENE 6 reagent (Roche Diagnostic Systems). Eighteen
hours after transfection, the cells were washed twice and subsequently
stimulated with different concentrations of recombinant flagellin (0 –1000
ng/ml) in the presence or absence of mouse IgG purified from vaccinatedmice for 6 h. Cells were then washed and lysed in Passive Lysis Buffer(Promega). Luciferase assays were performed using a Dual-Luciferase Reporter Assay System (Promega) and analyzed with a luminometer (Veritas;
Promega).

FIGURE 2. Immunogenicity of flagellin DNA vaccines. A, Cross-reactivity of IgG raised by FliC WT, R90A, or FlaA was examined by immunoblot analysis using flagella prepared from PAO1 or PAK endogenously expressing FliC or FlaA, respectively. For PAO1 blots, the proximal bandcorresponds to the size estimated from the amino acid sequence, whereas distal one was supposed to be a degraded product. B–E, BALB/c mice were
immunized twice (0 and 3 wk) with either 50 ␮g of pGACAG (vector), pGACAG-FliC WT, pGACAG-FliC R90A, or pGACAG-FlaA via i.m.
electroporation. Blood was drawn 6 wk after the first immunization. Serum IgG titers against FlaA (C) or FliC (B) purified from PAK or PAO1,
respectively, were determined by ELISA. The graph shows the mean ⫾ SD, n ⫽ 8 mice/group; ⴱ, p ⬍ 0.05 by Student’s t test. Three independent
experiments gave similar results.

1150

FLAGELLIN IMMUNIZATION BLOCKS INNATE IMMUNE RESPONSESFIGURE 3. Protective potential of flagellin DNA vaccines. Mice were immunized as described in Fig. 2. A and B, Survival rate was monitored following
intranasal challenge with 2 LD50 doses of PAO1 or PAK. Three independent experiments gave similar results; ⴱ, p ⬍ 0.05 by Mantel-Cox log rank test.
C and D, Two weeks after final immunization, mice were challenged intranasally with 5 ⫻ 105 CFU of PAO1 or PAK. Twenty-four hours p.i., mice were

The Journal of Immunology

1151

FIGURE 4. Type-specific antiflagellin Ab inhibits the TLR5 activation by homologous, but not heterologous, type of flagellin. A–D, HEK293
cells were transiently transfected as described in Fig. 1. The cells were stimulated with different concentrations ofeither recombinant FliC or FlaA in thepresence of either 10 ␮g/ml control, antiFliC WT, FliC R90A, or FlaA IgG, andluciferase assay was performed. Three
independent experiments gave similar
results; ⴱ, p ⬍ 0.01 by Student’s t test.
E, Two micrograms of rFliC waspreincubated with 10 ␮g of antiFliC WT or R90A IgG at 37°C for
30 min. BALB/c mice were inoculated intranasally with this mixture.
Four hours after inoculation, mice
were challenged with 2 LD50 dosesof PAO1, and the survival rate wasmonitored for the next 10 days, n ⫽10 mice/group; ⴱ, p ⬍ 0.01 by Mantel-Cox log rank test. Similar results
were obtained in three independent
experiments.

Cytoprotective assay
The cytoprotective effect of flagellin was performed according to themethod described previously, with a little modification (4). Six-week-old
female BALB/c mice were used. Purified flagellin (2 ␮g) was preincubatedwith 10 ␮g of each purified IgG at 37°C for 30 min. The mice (eight mice
per group) were then inoculated intranasally with this mixture. Four hours
after inoculation, the mice were challenged with 2 LD50 doses of P. aeruginosa PAO1, and mortality was monitored for the subsequent 10 days.

Statistical analysis
ANOVA Bonferroni multiple comparison test and Mantel-Cox log rank
test were used for the analysis of data. A value of p ⬍ 0.05 was considered
as statistically significant.

Results
Characterization of flagellin FliC mutants for their TLR5
engagement
To examine the contribution of TLR5 activation by flagellin to its
immunogenicity and protective potential against P. aeruginosa infection, TLR5 activation-null mutants (inactive flagellin) were designed. Previous studies showed that TLR5 activation by the FliC
of S. typhimurium or FlaA of P. aeruginosa is mediated by a domain composed of the NH2- and COOH-terminal regions forming
several ␣ helices (7, 9). Based on this information, five Pseudo-monas FliC mutants were generated by replacing a single aminoacid in each ␣ helical region with an alanine (Fig. 1A). Each recombinant protein was purified under endotoxin-free conditions
and characterized for its ability to trigger through TLR5. HEK293
cells were transfected with a mouse or human TLR5 expression
plasmid plus an NF-␬B-dependent luciferase reporter plasmid.
These cells responded to stimulation with full-length flagellin, but
not with LPS, peptidoglycan, or recombinant GST (Fig. 1, B andC, and data not shown). Of interest, the mutagenized L88A and
R90A mutants triggered TLR5-dependent NF-␬B activation⬎100-fold less efficiently than WT FliC ( p ⬍ 0.01).
The biological activity of these flagellar proteins was then examined. Intranasal inoculation of recombinant FliC WT, FliC
Q97A, FliC V404A, FliC F425A, or FlaA WT strongly up-regulated the pulmonary production of TNF-␣ (Fig. 1D). By comparison, the level of TNF-␣ in bronchoalveolar lavage fluids frommice treated with FliC L88A or FliC R90A was significantly reduced, suggesting that these mutants are inactive flagellin for
TLR5-mediated proinflammatory responses ( p ⬍ 0.05; Fig. 1D).
To determine whether these mutants retained their antigenic
epitopes, the ability to bind IgG Abs raised against FliC was examined by ELISA. Results indicate that anti-flagellin Abs boundequally well to FliC WT and R90A, but not to L88A. Anti-FliC

sacrificed and the lung was removed. C and D, The number of live bacteria in the lung homogenate was enumerated. The graph shows the mean ⫾ SD,n ⫽ 5 mice/group. Similar results were obtained in three independent experiments; ⴱ, p ⬍ 0.0001 by Student’s t test. E, MPO activity in the lunghomogenates was determined at 24 h following intranasal challenge with PAO1. F, Lung tissue inflammation was analyzed at 24 h following intranasal
challenge with PAO1. The lung sections were stained with H&E. Bar, 100 ␮m (left) and 50 ␮m (right), respectively. The graph shows the mean ⫾ SD
values, n ⫽ 5 mice/group; ⴱ, p ⬍ 0.05 by Student’s t test. Similar results were obtained in three independent experiments.

1152

FLAGELLIN IMMUNIZATION BLOCKS INNATE IMMUNE RESPONSESFIGURE 5. TLR5 plays crucial roles in the host’s bactericidal activity against P. aeruginosa. TLR5-deficient (TLR5⫺/⫺, blue) or control (TLR5⫹/⫺,
black) mice were challenged with 0.5 LD50 (A and C) or 1.5 LD50 (B) doses of the P. aeruginosa strain PAO1. A and B, Survival rate was monitored for
10 days following intranasal challenge. C, Twelve or 24 h p.i., mice were sacrificed and the lungs were removed, n ⫽ 10 mice/group. The number of live
bacteria in the lung homogenate was enumerated. The graph shows the mean ⫾ SD; ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 by Student’s t test. Two independent
experiments gave similar results.

serum also cross-reacted with recombinant FlaA, but with lower
affinity (Fig. 1E). These findings suggest that FliC R90A, but not
L88A, retained the antigenic conformation of flagellin. Taken together, these findings indicate that FliC R90A is antigenic but triggers via TLR5 less well than WT flagellar proteins (inactive
flagellin).
Immunogenicity of DNA vaccines encoding active or inactive
flagellin (FliC WT, R90A, or FlaA)
Mice were immunized and boosted with DNA vaccines encoding
active or inactive flagellin (FliC WT, FliC R90A, or FlaA). Sera
from these animals were studied for reactivity with flagellar proteins from the PAO1 and PAK strains of P. aeruginosa (expressing
FliC and FlaA, respectively). Sera from unvaccinated mice did notreact with either protein (data not shown). Sera from all of theimmunized mice reacted with both types of flagella, suggestingthat each vaccine induced Abs capable of cross-reacting with bothtypes of flagellin (Fig. 2A). Of interest, serum raised against
FlaA reacted better to the degraded or an alternative form (Fig.
2A, lower band) than full-length FliC (Fig. 2A, upper band).
This may reflect recognition of some cryptic epitopes of FliC by
anti-FlaA Abs.
The magnitude of each serum IgG response was quantitated by
ELISA (Fig. 2, B and C). After boosting, significant IgG antiPAO1 (FliC) and anti-PAK flagella (FlaA) responses were present
in all vaccination groups. The FliC R90A DNA vaccine induced
the weaker IgG responses against both types of flagella comparedwith those by FliC WT vaccine ( p ⬍ 0.05; Fig. 2, B and C),
suggesting either that the mutation introduced into R90A affected
its immunogenicity or that TLR5-mediated activation of the innate
immune system contributed to maximizing the anti-flagellar response elicited by vaccines encoding WT flagellin.
Protective activity of DNA vaccines targeting flagellin
The capacity of each DNA vaccine to protect against P. aeruginosa infection was assessed by challenging mice intranasally with
the PAO1 or PAK strain of bacteria (Fig. 3, A and B). Unexpectedly, mice vaccinated with FlaA were better protected against
PAO1 (which expresses FliC) than PAK (which expresses FlaA),
whereas mice vaccinated with the FliC DNA vaccine were better
protected against PAK than PAO1. These findings suggest thateach flagellar vaccine elicited stronger protection against bacteria
expressing the heterologous type of flagellin. In contrast, the DNA
vaccine targeting the inactive flagellin FliC R90A induced strong
protection against both strains of P. aeruginosa (Fig. 3, A and B).

The effect of each DNA vaccine on host susceptibility to bacterial infection was further evaluated by examining bacterial persistence and neutrophil recruitment in the lung following intranasal
infection with a sublethal dose of P. aeruginosa. Consistent withdata from the protection studies, mice immunized with DNA vaccines encoding FlaA or FliC R90A efficiently eliminated PAO1,
whereas mice immunized with the FliC WT or R90A vaccines
efficiently eliminated PAK (Fig. 3, C and D). When neutrophil
recruitment was examined by monitoring MPO activity, both FliC
R90A and FlaA vaccines elicited stronger MPO activity than FliC
WT following challenge with PAO1 (Fig. 3E). The lung inflammation associated with P. aeruginosa infection was also assessedhistopathologically. When mice were challenged with PAO1, thelung inflammation was characterized by massive neutrophil infiltration among mice vaccinated with FliC R90A or FlaA relative tothose vaccinated with vector or FliC WT (Fig. 3F). Taken together,
these findings raise the interesting possibility that the presence ofa homologous TLR5 activation domain in a flagellin-based vaccine
hampers the induction of protective inflammatory responses
against homologous, but not heterologous, strains of bacteria.

Anti-flagellin Ab blocks TLR5 activation in a type-specificmannerTo clarify the mechanism underlying the lower protection against
bacteria-expressing flagellin homologous to that present in the vaccine, sera from immunized mice were analyzed for their ability toblock TLR5 activation. Recombinant flagellin dose-dependently
stimulated both mouse and human TLR5 in the presence of control
IgG (Fig. 4, A–D). Anti-FliC WT, but neither FliC R90A nor FlaA
IgG, significantly suppressed the activation by a higher concentration of rFliC (1000 ng/ml, p ⬍ 0.001; Fig. 4A). A similar result
was observed when human TLR5 activation was examined (Fig.
4B). Anti-FlaA IgG specifically suppressed both mouse and human
TLR5 activation by rFlaA ( p ⬍ 0.01; Fig. 4, C and D). Combinedwith data from the protection studies, these results suggest thatanti-flagellin Abs induced against the domain responsible for
TLR5 activation hampered the host’s ability to generate an innate
immune response against P. aeruginosa, thereby increasing susceptibility to infection. It was also noted that serum from mice
immunized with the FliC DNA vaccine weakly but significantly
stimulated mouse TLR5. This may reflect epitope mimicry between FliC and the Id of the anti-FliC Ab, as reported in previous
studies (37).

The Journal of Immunology
Anti-flagellin Ab inhibits host protection against P. aeruginosa
infection
To evaluate the contribution of anti-FliC Ab response to host protection against P. aeruginosa, mice were inoculated intranasallywith rFliC in the presence of anti-FliC WT or FliC R90A IgG andthen challenged with the PAO1 strain of bacteria (Fig. 4E). Pretreatment with 2 ␮g of rFliC for 4 h conferred protection against
2 LD50 doses of intranasal challenge with bacteria (data notshown). This protection was blocked when rFliC was preincubatedwith anti-FliC WT IgG. However, anti-FliC R90A IgG had weakerpotential for the blocking of the rFliC-mediated protective effect( p ⬍ 0.01; Fig. 4E). This observation suggests that inactive flagellin FliC R90A vaccine raises a weaker level of blocking Ab production, which tolerates a rFliC-mediated anti-Pseudomonas innate immune response required for the elimination of bacteria.
TLR5 activation contributes to protection against P. aeruginosa
infection
The ability of flagellin to elicit a protective innate immune response through its interaction with TLR5 was further investigated(Fig. 5). TLR5-deficient (TLR5⫺/⫺) and control (TLR5⫹/⫺) mice
were challenged intranasally with 0.5 or 1.5 LD50 doses of
Pseudomonas. The TLR5-deficient mice were significantly more
susceptible to challenge with 0.5, but not with 1.5, LD50 doses thanwere the controls ( p ⬍ 0.05; Fig. 5, A and B). When challenged
with sublethal doses of Pseudomonas, the number of infectious
bacteria persisting in the lungs was higher in TLR5-deficient thancontrol mice, suggesting that bactericidal activity against P.
aeruginosa is impaired in TLR5-deficient mice ( p ⬍ 0.01; Fig.
5C). These findings are consistent with flagellin-mediated TLR5
activation improving host resistance to infection by flagellated
bacteria (20).

Discussion
The activation of TLR5 by flagellin has an adjuvant-like effect thatenhances the immunogenicity of epitopes expressed on the flagellin molecule (13, 20). However, Abs generated against the TLR5
activation domains expressed by WT flagellin hamper the host’s
ability to mount a protective innate immune response against flagellated pathogens. Indeed, although vaccination with WT flagellin
induces a strong Ag-specific Ab response, it does not reduce hostsusceptibility to infection by bacteria expressing homologous
TLR5 activation domains. This contrasts to the effect of the mutant
flagellin FliC R90A, which interacts poorly with TLR5 and, thus,
induces only minimal levels of anti-TLR5-blocking Abs. Vaccination with FliC R90A provided protection against bacteria expressing either homologous or heterologous types of flagellin.
Studies have been initiated to examine the breadth of the protection conferred by flagellin FliC R90A against a wide variety of P.
aeruginosa strains.
The magnitude of the Ab response induced by flagellin is significantly improved by activation of the innate immune system.
Sanders et al. (38) demonstrated that Ab production following Salmonella rFliC immunization was severely impaired in MyD88
knockout mice. Helicobacter pylori FlaA, which interacts poorly
with TLR5 and, thus, induces only weak anti-FlaA Ab, becomes
highly immunogenic when coadministered with CFA (38). Our
results are consistent with these observations. The inactive
Pseudomonas flagellin FliC R90A induced significantly lower levels of Ab production when compared with active Pseudomonas
flagellin FliC or FlaA (Fig. 2, B and C). Thus, TLR5- and MyD88mediated innate immune activation influences the magnitude of theanti-flagellin Ab response. However, this response includes both1153
protective anti-flagellin Abs and Abs that neutralizing TLR5
activity.
P. aeruginosa possesses another major pathogen-associated molecular pattern, LPS, which is the potent activator of the host’s
defensive innate immune responses (39). However, previous studies have demonstrated that lung epithelial cells poorly recognize
LPS (40, 41). Results from the present study suggest that flagellin
may be the dominant inflammatory trigger of airway epithelia(Fig. 5). Yet, it should be noted that we observed no significant
difference between TLR5-deficient and its counterpart control
mice in their resistance to relatively high challenge doses of P.
aeruginosa (42).
Several classes of flagellin-mediated protection mechanisms
have been implicated in the host’s mucosal immunity. Molofsky
et al. (3) demonstrated that aflagellated Legionella persists longer
than WT bacteria following pulmonary infection, in part due to theapoptosis of lung epithelial cells. Aflagellated Salmonella elicitedsevere mucosal damage by a mechanism that also appears to involve the induction of apoptosis in gut epithelia via inhibition of
NF-␬B activation (4). Thus, certain bacterial flagellin may modulate host’s mucosal inflammation by triggering NF-␬B and altering
apoptosis signaling in epithelial cells. Blocking the TLR5 activation domain of flagellin may therefore permit bacteria to evade thehost’s innate immune responses and alter the level of tissue damage associated with late-stage bacterial infection.
Many studies have demonstrated that pathogenic bacteria possess versatile systems to evade from the host’s immune surveillance. For instance, Yersinia pestis produces tetra-acylated LPS,
which acts as an antagonist to TLR4, preventing the induction of
TLR4-mediated inflammatory responses upon infection. After colonization, P. aeruginosa down-regulates the expression levels offlagella to evade from the TLR5-mediated host’s innate immune
activation (43). H. pylori may have evolved to express inactive
flagellin to prevent activation of the TLR5-mediated host’s mucosal immunity, enabling persistent infection in the stomach epithelia(6). Current data provide another mechanism by which bacteria
can subvert host induction of an innate immune response.
In summary, these studies are the first to demonstrate that Ab
responses directed against the TLR5 activation domain of flagellin
actually hamper the induction of protective immunity. By modifying flagellin to prevent this Ab response, the host’s ability tomount protective innate immune responses against flagellated
pathogens is significantly improved. These observations have important implications for vaccine development against flagellated
pathogens.

Acknowledgments
We thank Dr. K. Ogata for discussion, A. de la Fuente for secretarial
assistance, and M. Tozuka and K. Takase for technical assistance.

Disclosures
The authors have no financial conflict of interest.

References
1. Shapiro, L. 1995. The bacterial flagellum: from genetic network to complex architecture. Cell 80: 525–527.
2. Pier, G. B., G. Meluleni, and J. B. Goldberg. 1995. Clearance of Pseudomonas
aeruginosa from the murine gastrointestinal tract is effectively mediated by Oantigen-specific circulating antibodies. Infect. Immun. 63: 2818 –2825.
3. Molofsky, A. B., B. G. Byrne, N. N. Whitfield, C. A. Madigan, E. T. Fuse,
K. Tateda, and M. S. Swanson. 2006. Cytosolic recognition of flagellin by mouse
macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203:
1093–1104.
4. Vijay-Kumar, M., H. Wu, R. Jones, G. Grant, B. Babbin, T. P. King, D. Kelly,
A. T. Gewirtz, and A. S. Neish. 2006. Flagellin suppresses epithelial apoptosis
and limits disease during enteric infection. Am. J. Pathol. 169: 1686 –1700.
5. Uematsu, S., M. H. Jang, N. Chevrier, Z. Guo, Y. Kumagai, M. Yamamoto,
H. Kato, N. Sougawa, H. Matsui, H. Kuwata, et al. 2006. Detection of pathogenic

1154

FLAGELLIN IMMUNIZATION BLOCKS INNATE IMMUNE RESPONSES

intestinal bacteria by Toll-like receptor 5 on intestinal CD11c⫹ lamina propria
cells. Nat. Immunol. 7: 868 – 874.
6. Andersen-Nissen, E., K. D. Smith, R. Bonneau, R. K. Strong, and A. Aderem.
2007. A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin.
J. Exp. Med. 204: 393– 403.
7. Smith, K. D., E. Andersen-Nissen, F. Hayashi, K. Strobe, M. A. Bergman,
S. L. Barrett, B. T. Cookson, and A. Aderem. 2003. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat. Immunol. 4: 1247–1253.
8. Murthy, K. G., A. Deb, S. Goonesekera, C. Szabo, and A. L. Salzman. 2004.
Identification of conserved domains in Salmonella muenchen flagellin that are
essential for its ability to activate TLR5 and to induce an inflammatory response
in vitro. J. Biol. Chem. 279: 5667–5675.
9. Verma, A., S. K. Arora, S. K. Kuravi, and R. Ramphal. 2005. Roles of specificamino acids in the N terminus of Pseudomonas aeruginosa flagellin and of flagellin glycosylation in the innate immune response. Infect. Immun. 73: 8237– 8246.
10. Newton, S. M., C. O. Jacob, and B. A. Stocker. 1989. Immune response tocholera toxin epitope inserted in Salmonella flagellin. Science 244: 70 –72.
11. Cuadros, C., F. J. Lopez-Hernandez, A. L. Dominguez, M. McClelland, and
J. Lustgarten. 2004. Flagellin fusion proteins as adjuvants or vaccines induce
specific immune responses. Infect. Immun. 72: 2810 –2816.
12. McSorley, S. J., B. D. Ehst, Y. Yu, and A. T. Gewirtz. 2002. Bacterial flagellin
is an effective adjuvant for CD4⫹ T cells in vivo. J. Immunol. 169: 3914 –3919.
13. Applequist, S. E., E. Rollman, M. D. Wareing, M. Liden, B. Rozell, J. Hinkula,and H. G. Ljunggren. 2005. Activation of innate immunity, inflammation, andpotentiation of DNA vaccination through mammalian expression of the TLR5
agonist flagellin. J. Immunol. 175: 3882–3891.
14. Mizel, S. B., A. N. Honko, M. A. Moors, P. S. Smith, and A. P. West. 2003.
Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes. J. Immunol. 170: 6217– 6223.
15. Yu, Y., H. Zeng, S. Lyons, A. Carlson, D. Merlin, A. S. Neish, and A. T. Gewirtz.
2003. TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via posttranscriptional mechanism. Am. J. Physiol. 285: G282–G290.
16. Gewirtz, A. T., T. A. Navas, S. Lyons, P. J. Godowski, and J. L. Madara. 2001.
Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce
epithelial proinflammatory gene expression. J. Immunol. 167: 1882–1885.
17. Liaudet, L., C. Szabo, O. V. Evgenov, K. G. Murthy, P. Pacher, L. Virag,J. G. Mabley, A. Marton, F. G. Soriano, M. Y. Kirov, et al. 2003. Flagellin from
Gram-negative bacteria is a potent mediator of acute pulmonary inflammation insepsis. Shock 19: 131–137.
18. Means, T. K., F. Hayashi, K. D. Smith, A. Aderem, and A. D. Luster. 2003. The
Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol. 170: 5165–5175.
19. Rhee, S. H., A. C. Keates, M. P. Moyer, and C. Pothoulakis. 2004. MEK is a key
modulator for TLR5-induced interleukin-8 and MIP3␣ gene expression in nontransformed human colonic epithelial cells. J. Biol. Chem. 279: 25179 –25188.
20. Lee, S. E., S. Y. Kim, B. C. Jeong, Y. R. Kim, S. J. Bae, O. S. Ahn, J. J. Lee,
H. C. Song, J. M. Kim, H. E. Choy, et al. 2006. A bacterial flagellin, Vibrio
vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity. Infect. Immun. 74: 694 –702.
21. Strindelius, L., M. Filler, and I. Sjoholm. 2004. Mucosal immunization withpurified flagellin from Salmonella induces systemic and mucosal immune responses in C3H/HeJ mice. Vaccine 22: 3797–3808.
22. Sbrogio-Almeida, M. E., and L. C. Ferreira. 2001. Flagellin expressed by live
Salmonella vaccine strains induces distinct antibody responses following delivery
via systemic or mucosal immunization routes. FEMS Immunol. Med. Microbiol.
30: 203–208.
23. Adriaensen, C., H. De Greve, J. Q. Tian, S. De Craeye, E. Gubbels, V. Eeckhaut,
F. Van Immerseel, R. Ducatelle, M. Kumar, and J. P. Hernalsteens. 2007. A live
Salmonella enterica serovar enteritidis vaccine allowing serological differentiation between vaccinated and infected animals. Infect. Immun. 75: 2461–2468.
24. Kodama, C., and H. Matsui. 2004. Salmonella flagellin is not a dominant protective antigen in oral immunization with attenuated live vaccine strains. Infect.
Immun. 72: 2449 –2451.

25. Lee, L. H., E. Burg III, S. Baqar, A. L. Bourgeois, D. H. Burr, C. P. Ewing,
T. J. Trust, and P. Guerry. 1999. Evaluation of a truncated recombinant flagellinsubunit vaccine against Campylobacter jejuni. Infect. Immun. 67: 5799 –5805.
26. Neville, L. F., Y. Barnea, O. Hammer-Munz, E. Gur, B. Kuzmenko,
H. Kahel-Raifer, R. Eren, A. Elkeles, K. G. Murthy, C. Szabo, et al. 2005. Antibodies raised against N⬘-terminal Pseudomonas aeruginosa flagellin prevent
mortality in lethal murine models of infection. Int. J. Mol. Med. 16: 165–171.
27. Lanyi, B. 1970. Serological properties of Pseudomonas aeruginosa: II. typespecific thermolabile (flagellar) antigens. Acta Microbiol. Acad. Sci. Hung. 17:
35– 48.
28. Fukushima, J., S. Yamamoto, K. Morihara, Y. Atsumi, H. Takeuchi,
S. Kawamoto, and K. Okuda. 1989. Structural gene and complete amino acidsequence of Pseudomonas aeruginosa IFO 3455 elastase. J. Bacteriol. 171:1698 –1704.
29. Saha, S., F. Takeshita, S. Sasaki, T. Matsuda, T. Tanaka, M. Tozuka, K. Takase,
T. Matsumoto, K. Okuda, N. Ishii, et al. 2006. Multivalent DNA vaccine protects
mice against pulmonary infection caused by Pseudomonas aeruginosa. Vaccine
24: 6240 – 6249.
30. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, andD. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG DNAinduced activation of human cells. J. Immunol. 167: 3555–3558.
31. Kawamoto, S., Y. Shibano, J. Fukushima, N. Ishii, K. Morihara, and K. Okuda.
1993. Site-directed mutagenesis of Glu-141 and His-223 in Pseudomonas aeruginosa elastase: catalytic activity, processing, and protective activity of the elastaseagainst Pseudomonas infection. Infect. Immun. 61: 1400 –1405.
32. Takeshita, F., T. Tanaka, T. Matsuda, M. Tozuka, K. Kobiyama, S. Saha,
K. Matsui, K. J. Ishii, C. Coban, S. Akira, et al. 2006. Toll-like receptor adaptor
molecules enhance DNA-raised adaptive immune responses against influenza andtumors through activation of innate immunity. J. Virol. 80: 6218 – 6224.
33. Price, B. M., J. Barten Legutki, D. R. Galloway, B. U. von Specht,
L. B. Gilleland, H. E. Gilleland, Jr., and J. Staczek. 2002. Enhancement of theprotective efficacy of an oprF DNA vaccine against Pseudomonas aeruginosa.
FEMS Immunol. Med. Microbiol. 33: 89 –99.
34. Koffler, H., and T. Kobayashi. 1957. Purification of flagella and flagellin withammonium sulfate. Arch. Biochem. Biophys. 67: 246 –248.
35. Schneider, T., and A. C. Issekutz. 1996. Quantitation of eosinophil and neutrophil
infiltration into rat lung by specific assays for eosinophil peroxidase and myeloperoxidase: application in a Brown Norway rat model of allergic pulmonary
inflammation. J. Immunol. Methods 198: 1–14.
36. Takeshita, F., K. J. Ishii, K. Kobiyama, Y. Kojima, C. Coban, S. Sasaki, N. Ishii,
D. M. Klinman, K. Okuda, S. Akira, and K. Suzuki. 2005. TRAF4 acts as asilencer in TLR-mediated signaling through the association with TRAF6 andTRIF. Eur. J. Immunol. 35: 2477–2485.
37. Fields, B. A., F. A. Goldbaum, X. Ysern, R. J. Poljak, and R. A. Mariuzza. 1995.
Molecular basis of antigen mimicry by an anti-idiotope. Nature 374: 739 –742.
38. Sanders, C. J., Y. Yu, D. A. Moore, III, I. R. Williams, and A. T. Gewirtz. 2006.
Humoral immune response to flagellin requires T cells and activation of innate
immunity. J. Immunol. 177: 2810 –2818.
39. Ernst, R. K., E. C. Yi, L. Guo, K. B. Lim, J. L. Burns, M. Hackett, andS. I. Miller. 1999. Specific lipopolysaccharide found in cystic fibrosis airway
Pseudomonas aeruginosa. Science 286: 1561–1565.
40. Hybiske, K., J. K. Ichikawa, V. Huang, S. J. Lory, and T. E. Machen. 2004.
Cystic fibrosis airway epithelial cell polarity and bacterial flagellin determine host
response to Pseudomonas aeruginosa. Cell Microbiol. 6: 49 – 63.
41. Zhang, Z., J. P. Louboutin, D. J. Weiner, J. B. Goldberg, and J. M. Wilson. 2005.
Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5. Infect. Immun. 73: 7151–7160.
42. Feuillet, V., S. Medjane, I. Mondor, O. Demaria, P. P. Pagni, J. E. Galan,
R. A. Flavell, and L. Alexopoulou. 2006. Involvement of Toll-like receptor 5 in
the recognition of flagellated bacteria. Proc. Natl. Acad. Sci. USA 103:
12487–12492.
43. Murray, T. S., M. Egan, and B. I. Kazmierczak. 2007. Pseudomonas aeruginosachronic colonization in cystic fibrosis patients. Curr. Opin. Pediatr. 19: 83– 88.