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Infect Immun. Jun 2009; 77(6): 2356–2366.
Published online Apr 6, 2009. doi:  10.1128/IAI.00054-09
PMCID: PMC2687341

A Fusion Protein Vaccine Containing OprF Epitope 8, OprI, and Type A and B Flagellins Promotes Enhanced Clearance of Nonmucoid Pseudomonas aeruginosa[down-pointing small open triangle]


Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this study was to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as the outer membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscular immunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significant antiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type B flagellins generated OprI-specific IgG. Immunization with a combination of OprF epitope 8 (OprF311-341), OprI, and flagellins elicited high-affinity IgG antibodies specific to flagellins, OprI, and OprF that individually promoted extensive deposition of C3 on P. aeruginosa. Although these antibodies exhibited potent antibody-dependent complement-mediated killing of nonmucoid bacteria, they were significantly less effective with mucoid isolates. Mice immunized with the OprF311-341-OprI-flagellin fusion had a significantly lower bacterial burden three days postchallenge and cleared the infection significantly faster than control mice. In addition, mice immunized with the OprF311-341-OprI-flagellin fusion had significantly less inflammation and lung damage throughout the infection than OprF-OprI-immunized mice. Based on our results, OprF311-341-OprI-flagellin fusion proteins have substantial potential as components of a vaccine against nonmucoid P. aeruginosa, which appears to be the phenotype of the bacterium that initially colonizes CF patients.

Cystic fibrosis (CF) is a hereditary disease that is linked to a defective CF transmembrane receptor (CFTR) (48). In CF patients, the presence of a defective CFTR protein leads to dehydrated mucosal surfaces and disruption of ion transport. In the initial stages of disease, CF patients are infected with Staphylococcus aureus and Haemophilus influenzae but eventually become infected with nonmucoid Pseudomonas aeruginosa, a gram-negative opportunistic pathogen that is the major cause of morbidity and mortality in these patients (5, 27, 28, 61). Following colonization, P. aeruginosa undergoes a mucoid conversion to an alginate-overexpressing phenotype that is associated with biofilm development and enhanced resistance to antibiotic therapy (28). CF is characterized by lung inflammation mediated, in part, by chronic P. aeruginosa infection. P. aeruginosa possesses numerous virulence factors that facilitate evasion of the immune system (15, 37, 43, 49). For example, P. aeruginosa secretes enzymes such as alkaline protease and elastase, which degrade complement components and thus limit the role of complement in the clearance of early pulmonary P. aeruginosa infections (16). The critical role of complement in the clearance of P. aeruginosa is evidenced by the observation that C3 and C5 knockout mice were unable to clear P. aeruginosa after challenge (40, 69). In addition, P. aeruginosa expresses lipopolysaccharide variants that interfere with C3b deposition (52).

Initial efforts to develop a P. aeruginosa vaccine focused primarily on lipopolysaccharide. Although vaccination with P. aeruginosa lipopolysaccharide was effective in several animal models and led to the production of highly opsonic antibodies, the efficacy in human trials was limited by antigenic diversity of O antigens among P. aeruginosa isolates (11). Since flagellin, OprI, and OprF exhibit conserved amino acid sequences, more recent studies have focused on these proteins as potential vaccine antigens (14, 26, 31, 67, 68).

P. aeruginosa possesses two types of flagellins, type A and type B, that differ in amino acid composition and length of the hypervariable region. P. aeruginosa flagellins have the unique property of being potent adjuvants as well as protective antigens (8, 32, 42, 50). Previous work has established flagellin as a potent adjuvant in mice (1, 3, 9, 10, 23, 33-35, 45, 53, 56) as well as cynomolgus and African green monkeys (24, 36). A phase III clinical trial of P. aeruginosa flagellins in CF patients demonstrated that the vaccine was well tolerated and caused a 30% reduction in the incidence of infection (12). In related studies, immunization with the OprI antigen of P. aeruginosa and an appropriate adjuvant elicited a protective response in mice that correlated with the titer of OprI-specific immunoglobulin G (IgG) (14). In addition, an adenovirus expressing epitope 8 (amino acids 311 to 341) of OprF (i.e., the OprF311-341 protein) provided protection against acute P. aeruginosa infection (67, 68). Several investigators have focused on a fusion peptide containing OprF and OprI as a potential vaccine candidate. Although large amounts of this protein were required for an optimal response, immunization with an OprF-OprI fusion protein resulted in a 95-fold increase in the 50% lethal dose for mice. A subsequent study in burn patients revealed that an OprF-OprI fusion protein was immunogenic and well tolerated (26, 31).

Although these experimental P. aeruginosa vaccines have shown promise in initial clinical trials, none have achieved the level of response required for protection against P. aeruginosa in CF patients. After a critical review of the literature, we have identified several features that are critical for an effective P. aeruginosa vaccine: the presence of a potent adjuvant, the ability to induce high-titer antigen-specific IgG that exhibits a high degree of functional activity (for example, complement activation), multivalency, and the ability to induce a robust memory response. To that end, we generated a multivalent vaccine containing type A and B flagellins, OprF, and OprI and have evaluated its immunogenicity and protective potential. A key feature of the vaccine is the presence of flagellin, a potent adjuvant that signals via Toll-like receptor 5 (TLR5).


Strains and plasmids.

Bacterial strains and plasmids used in this study are described in Table Table1.1. Escherichia coli cultures were maintained at 37°C in Luria-Bertani (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) broth, while P. aeruginosa was cultured in LB broth lacking NaCl (LBNS) (10 g/liter tryptone, 5 g/liter yeast extract). Solid media were prepared by adding 1.0 to 1.5% Select agar (Gibco-BRL). Plasmids in E. coli were selected using media supplemented with antibiotics (carbenicillin, 100 μg ml−1; gentamicin, 10 μg ml−1). Plasmids in P. aeruginosa were selected on media containing carbenicillin (300 μg ml−1), gentamicin (100 μg ml−1), and Irgasan (25 μg ml−1). E. coli strain JM109 was used for all cloning procedures, while E. coli SM10 was used to transfer plasmids into P. aeruginosa by biparental mating (60). The P. aeruginosa strains used were PAO1 and its derivatives WFPA850, WFPA852, WFPA854, WFPA860, WFPA862, WFPA864, and WFPA866. Vectors pEX18Gm and pEX18Ap or derivatives were used for cloning and gene replacements (Table (Table11).

Bacterial strains used in this study

Construction of nonpolar fliC, oprF, and oprI deletion mutations.

To engineer unmarked, nonpolar fliC, oprF, and oprI deletion mutations, we utilized a previously described method (57). Internal fragments of coding sequences within each gene were deleted using a modified PCR technique termed splicing by overlap extension (65). In this assay, four gene-specific primers were employed in three separate PCRs to generate DNA fragments with a defined in-frame deletion of coding sequences within the fliC, oprF, or oprI genes. The primers were also designed such that the final amplicon, harboring the specified mutated allele harbored restriction sites to allow direct cloning into pEX18Ap or pEX18Gm, resulting in plasmid pHL150 (ΔfliC), pHL153 (ΔoprF), or pHL155 (ΔoprI). The mutant alleles were introduced into the PAO1 chromosome as outlined previously (21). The merodiploids were resolved by growing on sucrose-containing media and introduction of the mutated allele, which was verified by PCR.

Recombinant proteins.

DNA encoding full-length type A flagellin of P. aeruginosa strain PAK and DNA encoding full-length type B flagellin of strain PAO1 were each amplified by PCR and ligated into pET29a. DNA encoding the mature OprI antigen of P. aeruginosa strain PAO1 (amino acids 21 to 83) was amplified by PCR and ligated into pET29a or to the 5′ end of type A and B flagellin genes in pET29a, generating constructs that encode OprI-type A flagellin and OprI-type B flagellin (OprI-flagellins). DNA encoding epitope 8 (amino acids 311 to 341) of OprF of P. aeruginosa strain PAO1 was amplified by PCR and ligated into pET29a or to the 5′ end of the OprI and type A flagellin gene construct and the OprI and type B flagellin gene construct. The structure of each final protein is presented in diagrammatic form in Fig. Fig.11.

FIG. 1.
Illustration of the constructs used in this study. Epi8, epitope 8.

All expressed proteins were purified by metal ion affinity chromatography as previously described (1, 24). Acrodisc Q membranes were used to deplete endotoxin and nucleic acids. Endotoxin levels were <10 pg/μg for all of the proteins (as detected by the QCL-1000 chromogenic Limulus amebocyte lysate test kit [Cambrex Corporation, East Rutherford, NJ]).

ELISA for TNF-α and antigen-specific IgG.

Tumor necrosis factor alpha (TNF-α) levels in cultures of RAW 424 (TLR5-positive [TLR5+]) or RAW 264.7 (TLR5-negative [TLR5]) cells were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (OptiEIA ELISA; Becton Dickinson) according to the manufacturer's instructions. Data represent three independent experiments with triplicate samples in each experiment.

Titers of antigen-specific IgG were measured using MaxiSorp plates coated with 100 μl of antigen (type A flagellin, type B flagellin, OprI, or OprF) at 10 μg/ml in sterile phosphate-buffered saline (PBS). The plates were incubated overnight at 4°C and then blocked with 10% newborn calf serum in PBS. Plasma samples (in triplicate) were added, and the plates were incubated overnight at 4°C, followed by secondary anti-Ig antibodies (Roche Diagnostics) for 2 h at room temperature. Peroxidase activity was detected with 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrate system (Sigma-Aldrich) and stopped with 2 N H2SO4. Endpoint dilution titers were defined as the inverse of the lowest dilution that resulted in an absorbance value (at 450 nm) 0.1 higher than that of naive plasma. Groups of at least seven mice were used. To determine relative antibody affinities, the ELISA was conducted as described above with the addition of a 15-min incubation with sodium thiocyanate (NaSCN) (Sigma) solution as described previously (1, 29).


Six- to 8-week-old BALB/c and DBA/2 mice were purchased from Charles River Laboratories. All animals were maintained under pathogen-free conditions. All research performed on mice in this study complied with federal and institutional guidelines set forth by the Wake Forest University Animal Care and Use Committee.

Intramuscular immunization of mice.

Groups of seven mice were anesthetized with 2,2,2-tribromoethanol (Avertin; Sigma) and tert-amyl alcohol (Fisher) by intraperitoneal injection. Small volumes (20 μl total) containing antigen and adjuvant in PBS were injected using a 29.5-gauge needle into the right calf of each mouse. Mice were boosted at 4 weeks via the same route and bled two weeks later. Plasma was prepared and stored at −70°C until analysis.

ELISPOT assay.

The frequency of antigen-specific plasma cells was determined using limiting dilution analysis as previously described (54). Briefly, Immobulin-P high-affinity protein binding enzyme-linked immunospot (ELISPOT) plates (Millipore) were coated with 100 μl of type A flagellin, type B flagellin, OprI, or OprF (10 μg/ml) in sterile PBS. Bone marrow (BM) and spleen were collected 45 days postboost, single-cell suspensions were prepared, and dilutions of the cells (5 × 105/well) were added to the antigen-coated wells. Plates were then incubated at 37°C for 5 h, washed, and probed with goat anti-mouse antibody (4°C overnight). Plates were developed using horseradish peroxidase-Avidin D diluted 1:1,000 (Southern Biotechnology) and 3-amino-9-ethylcardbazole (AEC) and dried overnight. Spots were enumerated using a dissecting microscope. Only wells that contained ≥4 spots were counted for analysis. Total spleen plasma cell numbers were calculated by multiplying the number of cells in the spleen by the number of spots per million spleen cells. Total BM plasma cell numbers were calculated in the same manner with an additional multiplication by 7.9 to compensate for total BM (2).

To determine the frequency of antigen-specific memory B cells (MBC), the BM and spleen cells were incubated in vitro for 5 days in the presence of 1 μg/ml OprF311-341-OprI-flagellins and then plated as described above. The number of MBC was determined by subtracting the number of plasma cells from the 5-h incubation from the total number of plasma cells after the 5-day culture. Results for two independent experiments are given.

Antigen-specific IgG binding to P. aeruginosa.

P. aeruginosa strains were incubated with heat-inactivated control or immune mouse plasma for 1 h at 4°C prior to staining with Alexa Fluor 647-conjugated anti-mouse IgG (Invitrogen) for 1 h at 4°C. Data are representative of two experiments with triplicate samples in each experiment.

Antigen-specific IgG-mediated complement activation.

Control and immune mouse plasma were diluted 1:10 and heat inactivated at 56°C for 1 h prior to use. P. aeruginosa strains were grown in LBNS broth to an optical density at 600 nm of 0.5 (~108 CFU/ml), washed two times with sterile PBS, and then incubated with mouse plasma for 1 h. The bacteria were then washed and incubated for 1 h at 37°C with 5% rabbit serum (Innovative Research) as a source of complement. Finally, the bacteria were stained with goat anti-rabbit C3-fluorescein isothiocyanate (MP Biomedical). Flow cytometric analysis was performed using a BD FACSCalibur, and data were analyzed with FloJo software (Tree Star, Inc., Ashland, OR). Histograms representative of results from three experiments are shown. Complement-mediated killing was performed as described above with the exception that the bacteria were incubated for 4 h with rabbit serum. A time course experiment revealed minimal killing at 1 h with rabbit serum (data not shown). The percentage of bacteria killed was quantitated by the following equation: (number of input bacteria − number of recovered bacteria)/(number of input bacteria) × 100.

Respiratory challenge with agar-embedded P. aeruginosa.

P. aeruginosa strains were grown in LB broth lacking NaCl to 108 CFU/ml. One part bacteria was added to nine parts warm (52°C) 1.5% Trypticase soy agar. After five minutes, the agar-bacterium mixture was injected into rapidly spinning warm heavy mineral oil by using a 22-gauge needle. The suspension was then mixed for 6 min. The agar beads were then cooled on ice for 20 min and washed three times with sterile PBS. The final volume was adjusted to approximately 5 ml. To determine the number of CFU/ml, the agar-bacterium beads were homogenized, and bead size was determined by comparison to 100- to 150-μm chromatography beads. Mice (six or seven per group) were anesthetized with 2,2,2-tribromoethanol (with tert-amyl alcohol) by intraperitoneal injection, and then 50 μl of agar-embedded bacteria was instilled intratracheally using a sterile gel-loading tip.


Lungs were harvested and transferred to 10% formalin for 24 h. The tissue was then trimmed, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin by routine methods. For histological examination, groups of four mice were used for each condition. Slides were blindly scored on an increasing severity index that incorporates values for consolidation, bronchiolar and vascular degenerative changes, and edema (range for each factor, 0 to 4). Total inflammation score was calculated by the sum of all categories. Representative images (see Fig. Fig.9)9) are shown from four animals/group, with three sections per animal.

FIG. 9.
OprF311-341-OprI-flagellin-immunized mice are protected against severe lung pathology during pulmonary P. aeruginosa challenge. The left lungs of identical mice used the experiments represented in Fig. Fig.77 were evaluated for histology. Lungs ...

Statistical analyses.

Statistical analysis was performed using SigmaStat 3.10 (Systat Software, Inc., Point Richmond, CA). For normally distributed data sets, significance was determined using Student's t test. The significances of data sets which were not normally distributed or were of unequal variances were determined using the Mann-Whitney rank sum test. Where applicable, a two-way analysis of variance test was applied. P values of less than 0.05 were considered significant.


TLR5-specific signaling activity of P. aeruginosa type A and B flagellins and OprF311-341-OprI-flagellins.

In order to generate antigens with flagellin as the adjuvant, we generated several constructs as shown in Fig. Fig.1.1. In view of the insertion of the OprF and OprI sequences at the N terminus of flagellin, it was important to determine if this addition would have a negative impact on the ability of each flagellin, i.e., type A or B, to signal via TLR5. To test the ability of P. aeruginosa flagellins either alone or as part of a three-part fusion with OprF and OprI (Fig. (Fig.1)1) to signal via TLR5, RAW 424 (TLR5+) or RAW 264.7 (TLR5) cells were incubated with 1 pM to 1 nM of each protein, and production of TNF-α was assessed. Stimulation of RAW 424 cells with P. aeruginosa type A or B flagellin, OprI and type A or B flagellin, or OprF311-341, OprI, and type A or B flagellin resulted in a concentration-dependent increase in TNF-α (Fig. 2A to C). In contrast, none of these proteins induced TNF-α production in cultures of TLR5 RAW 264.7 cells. Consistent with previous results with the P. aeruginosa flagellins, the half-maximal stimulation occurred at 16 pM for type A flagellin and 40 pM for type B flagellin (8). There was no significant difference between the half-maximal stimulations of type A or B flagellin, OprI and type A or B flagellin, or OprF311-341, OprI, and type A or B flagellin. Thus, the presence of OprF-OprI at the N terminus of type A or B flagellin does not alter recognition and signaling via TLR5.

FIG. 2.
TLR5-specific signaling activity of P. aeruginosa type A and B flagellins and OprF311-341-OprI-flagellin. RAW 424 (TLR5+) and RAW 264.7 (TLR5) cells were stimulated with 10−9 to 10−12 M of protein. At 4 h poststimulation, ...

Immunization with OprF311-341-OprI-flagellins promotes a potent antigen-specific humoral response.

To assess the ability of OprF311-341-OprI-flagellins to promote an antigen-specific humoral response, groups of seven BALB/c or DBA/2 mice were immunized with 5 μg of each flagellin (types A and B) plus 10 μg OprI, 5 μg OprI-flagellin fusion proteins, or 5 μg OprF311-341-OprI-flagellin fusion proteins. Prior experiments established that immunization of BALB/c mice with 5 μg OprI-flagellins generated a maximal IgG response to flagellin and OprI (data not shown). Control mice received either OprI or OprF311-341-OprI at equivalent molar doses. DBA/2 mice were used because previous studies identified DBA/2 mice as more susceptible to P. aeruginosa infection than BALB/c and C57BL/6 mice (55, 58). Four weeks later, mice were boosted in an identical manner. Two weeks after the boost, the mice were bled and plasma was prepared for analysis of circulating antigen-specific IgG. Mice immunized with OprI-flagellins or OprF311-341-OprI-flagellins exhibited a robust OprI-specific IgG response (Fig. (Fig.3).3). In contrast, there was no significant OprI-specific IgG response in mice given only OprI or type A and B flagellins with OprI. In all cases, flagellin-specific responses were extremely robust. Mice immunized with OprF311-341-OprI-flagellins exhibited a high level of OprF-specific IgG as well as flagellin- and OprI-specific IgG.

FIG. 3.
Immunization with OprF311-341-OprI-flagellin promotes a potent antigen-specific humoral response. BALB/c or DBA/2 mice were immunized intramuscularly with 5 μg of type A and B flagellins plus 10 μg OprI, 5 μg OprI-type A flagellin ...

In addition to determining the titers of antigen-specific IgG following immunization with OprF311-341-OprI-flagellins, we also evaluated IgG isotypes and IgE. Plasma was prepared from immune mice as described above, and antigen-specific IgG subclasses and IgE were determined by ELISA. Immunization of mice with OprF311-341-OprI-flagellins did not elicit any detectable antigen-specific IgE (data not shown). This finding is consistent with our prior work demonstrating that flagellin does not promote antigen-specific IgE responses (24). Although high titers of antigen-specific IgG2a were induced, the overall response to OprI-flagellins or OprF311-341-OprI-flagellins was biased toward IgG1 (data not shown). This finding is consistent with our prior work on flagellin as an adjuvant in a Yersinia pestis vaccine (24).

Generation of antigen-specific plasma cells and MBC in response to OprF311-341-OprI-flagellins.

In view of the robust antigen-specific IgG response, we evaluated the frequency of antigen-specific plasma cells and MBC generated in response to OprF311-341-OprI-flagellins. Mice were immunized with 5 μg of OprF311-341-OprI-flagellins as described above, and 45 days postboost, BM and spleens were harvested and the frequencies of antigen-specific plasma cells and MBC were determined by ELISPOT assay. Antigen-specific plasma cells were determined following 5 h incubation. Eighty-five percent of antigen-specific plasma cells were found in the BM, and 15% were found in the spleen. Consistent with the IgG titer data (Fig. (Fig.3),3), there were more plasma cells for type A and B flagellins (~200/106 BM cells) than for OprI (42) and OprF (30) (Fig. (Fig.4A).4A). No plasma cells were detected in wells that contained cells from nonimmune mice. Although significantly more antigen-specific plasma cells were found in the BM, a substantial number of plasma cells remained in the spleen (Fig. (Fig.4B).4B). The retention of antigen-specific plasma cells in the spleen correlated with the immunogenicity of each antigen.

FIG. 4.
Generation of antigen-specific plasma cells and MBC by OprF311-341-OprI-flagellin immunization. DBA/2 mice were immunized intramuscularly with 5 μg of OprF311-341-OprI-flagellins. BM and spleens were harvested 40 days postboost and analyzed for ...

In contrast to the case for plasma cells, the generation of MBC specific to flagellins and the generation of MBC specific to OprI were equivalent (Fig. (Fig.4A).4A). The lower number of OprF-specific MBC (108 MBC/106 cells) was not unexpected, given the presence of only a single epitope. Nonetheless, our results clearly establish that OprF311-341-OprI-flagellins elicits not only significant numbers of plasma cells but also a substantial pool of MBC.

OprF311-341-OprI-flagellin immunization generates high-affinity antigen-specific IgG.

Since antigen affinity plays a critical role in the functional activity of an antibody, we evaluated the relative affinity of the IgG generated following immunization with OprF311-341-OprI-flagellins. The relative affinity of antibodies can be assessed in an ELISA by determining the concentration of sodium thiocyanate required to reduce antibody binding by 50%. As shown in Fig. Fig.5,5, immunization with OprI-flagellins or OprF311-341-OprI-flagellins generated IgG with equivalent relative affinities for the three antigens. For comparative purposes, a vaccine consisting of flagellin plus Y. pestis F1 antigen generated F1-specific IgG requiring 3 M sodium thiocyanate for 50% reduction in binding (1). Given the observation that these antibodies provide complete protection against respiratory challenge with Y. pestis (24, 36), we defined high-affinity IgG as antibodies requiring 2 to 3 M sodium thiocyanate for 50% reduction in antigen binding. OprF311-341-OprI-flagellin immune plasma had an average relative IgG affinity approaching 3 M for flagellins, OprI, and OprF (Fig. (Fig.5).5). Thus, the data are consistent with the conclusion that OprF311-341-OprI-flagellins elicits high-affinity antigen-specific IgG.

FIG. 5.
OprF311-341-OprI-flagellin immunization generates high-affinity antigen-specific IgG. Plasma samples from mice that received OprI-flagellins or OprF-OprI-flagellins were used to determine relative antibody affinities for type A flagellin, type B flagellin, ...

Complement-activating activity of antibodies specific for OprI, OprF, and type A and B flagellins.

To assess the functional activity of each of the antigen-specific IgG types, it was first necessary to generate P. aeruginosa mutants lacking one or more of these antigens (Table (Table1;1; see also Materials and Methods). The type B flagellin-expressing P. aeruginosa strain PAO1 was used as the genetic background for the mutants. Each mutant exhibited growth kinetics that were similar to that of the wild-type strain (data not shown). P. aeruginosa strains were incubated with immune or control mouse plasma at 4°C for 1 h and then stained for the presence of IgG. As shown in Fig. Fig.6A,6A, wild-type P. aeruginosa bound significant amounts of IgG specific for flagellin, OprI, and OprF. Furthermore, mutants positive for only flagellin, OprF, or OprI also bound high levels of IgG. Experiments using a type A flagellin-expressing strain, PAK, demonstrated similar results (data not shown). These results demonstrate that the antibodies generated against the recombinant fusion protein recognize these antigens in their cell-associated forms. This is particularly important in the case of OprF, since only a single epitope was present in the OprF311-341-OprI-flagellin fusion proteins.

FIG. 6.
Complement-activating activity of OprI, OprF, and type A and B flagellin-specific IgG antibodies. Plasma samples from OprF311-341-OprI-flagellin-immunized mice were incubated with P. aeruginosa, and IgG binding and C3 deposition was determined by flow ...

Having established the ability of the individual populations of IgG to recognize the cell-associated antigens, we next evaluated the potential of these antibodies to activate complement. Previous work has clearly established the importance of the complement system in the clearance of P. aeruginosa (13, 40, 52, 69). To assess the ability of IgG antibodies specific for OprF, OprI, and type A and B flagellins to activate complement, we measured the extent of IgG-dependent C3 deposition on P. aeruginosa. The various P. aeruginosa strains were incubated with a 1:10 dilution of heat-inactivated immune mouse plasma for 1 h, and then 5% rabbit complement was added for an additional hour. The bacteria were then stained with fluorescein isothiocyanate-labeled C3-specific antibody, and the extent of C3 deposition was determined by flow cytometry. A time course revealed that 1-h incubation with serum was optimal for C3 deposition and yielded minimal cell death (data not shown). As a control, we used a P. aeruginosa strain lacking flagellin, OprI, and OprF. OprI-flagellin or OprF311-341-OprI-flagellin immune plasma promoted significant C3 deposition on the surface of wild-type P. aeruginosa (Fig. (Fig.6A).6A). By using mutants that lack one or more of the eliciting antigens, we found that IgG with specificity for each of the eliciting antigens promoted robust C3 deposition (Fig. 6B and C). When all three antigens were present, there was a synergistic increase in the level of C3 deposition. These results indicate that OprF311-341-OprI-flagellin immunization generated antigen-specific IgG that exhibited a high degree of functional activity and that the combination of flagellin-, OprI-, and OprF-specific IgG antibodies triggered the highest level of C3 deposition.

Antibody-dependent complement-mediated killing of P. aeruginosa by OprF311-341-OprI-flagellin immune plasma.

In view of the robust ability of OprF-, OprI-, and type A and B flagellin-specific IgG antibodies to promote C3 deposition, we next examined the ability of those antibodies to promote complement-mediated killing of P. aeruginosa. Bacteria were incubated with heat-inactivated immune plasma for 1 h, and then 5% rabbit complement was added for an additional 4 h at 37°C. It is important to note that, like wild-type bacteria, the P. aeruginosa mutants were not susceptible to significant nonspecific killing by normal serum (data not shown). Approximately 90% of wild-type, nonmucoid P. aeruginosa (PAO1, PAK, and 1286) isolates as well as strains expressing type B flagellin, OprI, or OprF were susceptible to antibody-dependent complement-mediated killing (Fig. (Fig.77 and Table Table2).2). In contrast, only 18% of mucoid P. aeruginosa (T68933 and PDO300M) isolates were susceptible to killing (Table (Table2).2). This result is not unexpected, given the presence of a large amount of alginate exopolysaccharide in the mucoid strains that would likely mask OprI and OprF. In support of this conclusion, we found that a strain of PAO1 (PDO300NM) deficient in alginate production (and thus nonmucoid) was quite sensitive to killing (Table (Table2).2). In addition, the generally applicable inverse relationship between flagella and alginate expression (59) would also limit the effectiveness of the flagellin-specific IgG. The antigen dependence of the killing was evidenced by the very low level of killing, with bacteria lacking all three of the eliciting antigens. When the source of complement was heat inactivated, only background levels of killing were observed. Taken together, these findings clearly demonstrate that the antibodies generated in response to OprF311-341-OprI-flagellins exhibit potent antigen binding, complement-activating activity, and killing of nonmucoid but not mucoid P. aeruginosa.

FIG. 7.
Antibody-dependent complement (Comp)-mediated killing of P. aeruginosa by OprF311-341-OprI-flagellin-immunized mouse plasma. Plasma samples from OprF311-341-OprI-flagellin-immunized mice were diluted 1:10 and heat inactivated at 56°C for 1 h. ...
Complement-mediated killing of additional P. aeruginosa strains

Enhanced clearance of P. aeruginosa in OprF311-341-OprI-flagellin-immunized mice.

Nonmucoid P. aeruginosa does not cause a chronic infection in healthy mice as it does in CF patients. If large doses of bacteria are used, the mice quickly succumb to bacteremia (E. T. Weimer, D. J. Wozniak, and S. B. Mizel, unpublished observations). With small doses, the mice rapidly clear the bacteria. In view of the lack of a suitable animal model that closely mimics the situation in CF patients, i.e., chronic infection, investigators have evaluated agar-embedded mucoid P. aeruginosa as a way to infect mice such that rapid septic shock is avoided and the time of infection is lengthened (25, 58). For example, Stevenson and colleagues (55, 58) used the agar bead model to demonstrate that DBA/2 mice were more susceptible to mucoid P. aeruginosa than were BALB/c or C57BL/6 mice. However, since the initial P. aeruginosa infection in CF patients is mediated by nonmucoid strains (5, 28, 61), we felt it was more appropriate to use nonmucoid bacteria in the agar bead model. Preliminary results revealed that unimmunized mice did not succumb when infected intratracheally with up to 3.5 × 106 CFU of nonmucoid P. aeruginosa embedded in agar beads, but the mice did exhibit substantial morbidity. In view of the finding that DBA/2 mice are more susceptible to P. aeruginosa, we used this strain to evaluate the ability of OprF311-341-OprI-flagellin immunization to promote enhanced clearance of P. aeruginosa embedded in agar. DBA/2 mice were immunized as described above and infected intratracheally with 3.5 × 106 CFU of agar-embedded PAO1. Lungs were harvested 1, 3, and 5 days postinfection, and bacteria were enumerated by serial dilutions on LBNS plates. One day after challenge, immunized mice displayed a marked decrease in bacterial burden compared to control mice (Fig. (Fig.8).8). After 3 days, five of six mice immunized with OprF311-341-OprI-flagellins had cleared the infection. In contrast, the control mice had large numbers of bacteria in the lungs. Although the control mice cleared the infection by day 5, our results clearly demonstrate that immunization with OprF311-341-OprI-flagellins had a dramatic effect on the rate of bacterial clearance. It is important to emphasize that the ability of mice immunized with OprF-OprI to clear the infection by day 5 reflects a limitation of this model and not the efficacy of the OprF311-341-OprI-flagellin vaccine.

FIG. 8.
OprF311-341-OprI-flagellin-immunized mice display enhanced rate of clearance following pulmonary P. aeruginosa challenge. DBA/2 mice were immunized twice with 5 μg of OprF311-341-OprI-flagellins and challenged intratracheally with 3.5 × ...

Reduced lung pathology following pulmonary P. aeruginosa challenge in OprF311-341-OprI-flagellin-immunized mice.

In addition to determining bacterial burden following challenge, we also evaluated the histopathology of lungs from mice immunized with OprF311-341-OprI-flagellins or OprF-OprI. Lungs were harvested 1, 3, and 5 days after P. aeruginosa challenge. One day after P. aeruginosa challenge, alveolar walls from OprF311-341-OprI-flagellin-immunized mice displayed slight thickening owing to congestion and increased numbers of inflammatory cells. In contrast, lungs from mice immunized with OprF-OprI developed bronchopneumonia, with airway-oriented neutrophils, edema, and abundant visible bacteria (Fig. (Fig.9A).9A). After 3 days, immune mice exhibited only minor inflammatory changes in the lung, whereas more severe pneumonia with diffuse consolidation was present in the control animals. After 5 days, the lungs of immune mice were normal, while those of the controls had thickened alveolar walls, a result of congestion and inflammatory cells (Fig. (Fig.9B).9B). In summary, mice immunized with OprF311-341-OprI-flagellins displayed minimal lung pathology which completely resolved by day 5 postchallenge. The absence of lung pathology in the immune mice not only demonstrates the efficacy of the vaccine in promoting bacterial clearance but also the ability of the vaccine to promote clearance without inducing secondary tissue damage. In striking contrast, mice immunized with OprF-OprI demonstrated severe pneumonia which only partially resolved by day 5 (Fig. (Fig.9A).9A). In conjunction with the results of in vitro experiments (Fig. (Fig.55 and and6),6), it is clear that OprF311-341-OprI-flagellin immunization promotes an adaptive immune response that promotes the generation of antigen-specific IgG that exhibits robust functional activity, facilitates rapid clearance, and prevents the development of severe pneumonia following P. aeruginosa infection.


The goal of this study was twofold: to establish a set of criteria for a vaccine against P. aeruginosa and then to develop and test the vaccine based on these criteria. Based on our results, we conclude that the OprF311-341-OprI-flagellin vaccine meets all of the proposed criteria: the vaccine contains flagellin, a potent adjuvant, is multivalent, generates high-titer antigen-specific IgG that exhibits a high degree of functional activity, generates a robust memory response, and enhances clearance of nonmucoid P. aeruginosa without secondary tissue damage. Although the antigen-specific IgG induced by this vaccine did not promote complement-mediated killing of mucoid P. aeruginosa, it is important to emphasize that longitudinal studies of CF patients have clearly demonstrated that the initial P. aeruginosa infection is mediated by nonmucoid bacteria (5, 28, 61). We wish to emphasize that although the OprF311-341-OprI-flagellin vaccine has many of the features of an efficacious vaccine, the evaluation of its potential for use in CF patients is limited at this time by the lack of a suitable animal model that closely mirrors the situation in CF patients.

In addition to promoting high-level antigen-specific IgG (Fig. (Fig.3),3), flagellin also promoted the generation of significant numbers of antigen-specific MBC (Fig. (Fig.4).4). In view of these observations, it is likely that the very limited reduction in the incidence of P. aeruginosa infections in CF patients immunized with sheared flagella (12) may be due to a low level of functional adjuvant activity of the material. Although the adjuvants Pam3Cys and Pam2Ser (19) as well as alum (63) were found to enhance the response to P. aeruginosa antigens, it appears that flagellin is far more effective, as evidenced by the dramatic differences in the amounts of antigen required and the resultant titers of antigen-specific IgG.

We have shown that multivalency not only promotes synergistic activity of individual antibodies in activating complement but also enhances vaccine coverage against nonmucoid P. aeruginosa strains (Fig. (Fig.6).6). The value of multivalency was also demonstrated by Saha et al. (51), who used multivalent DNA vaccination with OprF-OprI, PilA, and PcrV antigens.

The titers of flagellin-, OprI-, and OprF-specific IgG antibodies following immunization with OprF311-341-OprI-flagellins are in most cases two logs higher than those reported in other studies (12, 14, 22, 50, 51, 67, 68, 70). For example, von Specht et al. (62) required three immunizations with 70-fold more antigen to achieve equivalent antibody responses. The difference may be due to the extraordinary potency of flagellin as an adjuvant, as well as the use of fusion proteins that enhance the efficiency of antigen delivery to dendritic cells via the binding of the associated flagellin to TLR5 on these cells (1a). The finding that immunization with OprF311-341-OprI-flagellins promotes the generation of large numbers of plasma cells is consistent with the very high titers of induced IgG. Furthermore, the generation of immunologic memory is evidenced by the relatively high frequency of antigen-specific MBC.

In line with prior work on flagellin, OprF, and OprI antigens, we have shown that all three antigens significantly impact the clearance of P. aeruginosa in mice. In addition, we have demonstrated that antibodies specific for each antigen activate complement and mediate killing of P. aeruginosa (Fig. (Fig.66 and and7).7). In this regard, von Specht and colleagues (13) found that the level of complement-activating OprI-specific IgG correlated with the level of protection against P. aeruginosa. Our results clearly support this conclusion. The finding that intramuscular immunization with OprF311-341-OprI-flagellins promotes clearance is consistent with the conclusion that protection is not dependent on the availability of antigen-specific IgA. In this regard, Pier et al. (44) demonstrated that intraperitoneal immunization could prevent mucosal colonization by P. aeruginosa.

A number of studies (4, 17, 38, 39) have presented evidence in support of the conclusion that heightened production of Th2 cell-derived cytokines, such as interleukin-4 (IL-4), IL-10, and IL-13, is associated with a poor prognosis in CF patients. In view of the notion that isotype switching to IgG1 is Th2 cell driven, the question arises as to whether the observed IgG1 bias (relative to IgG2a) of the humoral response to OprF311-341-OprI-flagellins might exacerbate the pathology in CF patients. First, the linkage between Th bias and prognosis in CF patients is based on cytokine production and not IgG isotype. Indeed, two studies revealed that elevated levels of alginate-specific IgG2 and IgG3, and not IgG1, are associated with a poor prognosis in CF patients (46, 47). It is also important to note that class switching to IgE is also promoted by Th2 cells, yet we have never observed any increase in IgE in response to OprF311-341-OprI-flagellins or any flagellin-based vaccine (24). Thus, we believe that the humoral response driven by the adjuvant activity of flagellin does not fit the paradigm of a classic Th2 response, and thus, the possibility that a flagellin-based P. aeruginosa vaccine might cause substantial secondary tissue damage is not warranted. This conclusion is clearly supported by the observation that the lungs of mice immunized with OprF311-341-OprI-flagellins did not exhibit any evidence of residual tissue damage following clearance of the bacteria (Fig. (Fig.99).

Although OprF311-341-OprI-flagellins promotes more rapid clearance of bacteria in healthy mice, it remains to be determined if it will be efficacious in CF patients. The pathophysiologic events that occur in CF patients as well as the virulence factors expressed by P. aeruginosa (66) clearly represent a significant challenge for any vaccine. The presence of a defective CFTR leads to abnormal ion transport and water depletion in the airway of CF patients that ultimately results in an increase in fluid viscosity, impaired mucociliary clearance, increased bacterial trapping in the mucus layer facilitating chronic infection, and a reduction in antibody penetration (18). In addition, infections by S. aureus and H. influenzae and later P. aeruginosa contribute to the development of a hyperinflammatory environment in the respiratory tract (7). All of these events contribute to a vicious cycle of infection and inflammation that ultimately causes severe pathology. Chronic inflammation can adversely affect vaccine efficacy due to the high-level production of prostaglandins that exert an inhibitory effect on lymphocytes (6). In addition, chronic inflammation promotes the continual recruitment of neutrophils to the lung that in turn release elastase that induces tissue damage and release of DNA, a substance that provides a scaffold for P. aeruginosa biofilms (20, 64).

The major cause of chronic inflammation in CF patients is persistent P. aeruginosa infection. Overproduction of alginate and, subsequently, biofilm development allow P. aeruginosa to evade the immune system and also increase antibiotic resistance, making it extremely difficult to eradicate the infection (5, 28). Mucoid conversion is also associated with a significant decrease in lung function in CF patients (41). Our findings indicate the best time to vaccinate CF patients using the OprF311-341-OprI-flagellin fusion would be prior to mucoid conversion, since complement-mediated killing occurred only in nonmucoid bacteria (Fig. (Fig.77 and Table Table2).2). Thus, the pathophysiologic events associated with CF in combination with the pathogenic mechanisms associated with P. aeruginosa represent a significant challenge to the elements of protective immunity. It is our view that vaccine efficacy will be most effective at a very early stage in the disease process. Perhaps the limited success of previous vaccine studies was not solely due to the limited inherent efficacy of the vaccines but rather to the timing of immunization.


We are grateful to John T. Bates, Kristen N. Delaney, and April B. Sprinkle for their excellent technical assistance.

This research was supported by a Pilot and Feasibility grant from the Cystic Fibrosis Foundation (MIZEL0810) and NIH grant AI061396 (to D.J.W.).


Editor: B. A. McCormick


[down-pointing small open triangle]Published ahead of print on 6 April 2009.


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