A Fusion Protein Vaccine Containing OprF Epitope 8, OprI, and Type

INFECTION AND IMMUNITY, June 2009, p. 2356–2366 Vol. 77, No. 60019-9567/09/$08.00�0 doi:10.1128/IAI.00054-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Fusion Protein Vaccine Containing OprF Epitope 8, OprI, and TypeA and B Flagellins Promotes Enhanced Clearance of Nonmucoid

Pseudomonas aeruginosa�

Eric T. Weimer,1 Haiping Lu,1 Nancy D. Kock,2 Daniel J. Wozniak,1 and Steven B. Mizel1*Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard,

Winston-Salem, North Carolina 27157,1 and Department of Pathology, Division of Comparative Medicine,Wake Forest University School of Medicine, Winston-Salem, North Carolina 271572

Received 15 January 2009/Returned for modification 20 February 2009/Accepted 26 March 2009

Although chronic Pseudomonas aeruginosa infection is the major cause of morbidity and mortality in cysticfibrosis (CF) patients, there is no approved vaccine for human use against P. aeruginosa. The goal of this studywas to establish whether a multivalent vaccine containing P. aeruginosa type A and B flagellins as well as theouter membrane proteins OprF and OprI would promote enhanced clearance of P. aeruginosa. Intramuscularimmunization with flagellins and OprI (separate) or OprI-flagellin fusion proteins generated significantantiflagellin immunoglobulin G (IgG) responses. However, only the fusions of OprI with type A and type Bflagellins 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 individuallypromoted 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 withmucoid isolates. Mice immunized with the OprF311-341–OprI–flagellin fusion had a significantly lower bacterialburden 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 lungdamage 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. aerugi-nosa, 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 adefective CF transmembrane receptor (CFTR) (48). In CFpatients, the presence of a defective CFTR protein leads todehydrated mucosal surfaces and disruption of ion transport.In the initial stages of disease, CF patients are infected withStaphylococcus aureus and Haemophilus influenzae but eventu-ally become infected with nonmucoid Pseudomonas aeruginosa,a gram-negative opportunistic pathogen that is the major causeof morbidity and mortality in these patients (5, 27, 28, 61).Following colonization, P. aeruginosa undergoes a mucoid con-version to an alginate-overexpressing phenotype that is asso-ciated with biofilm development and enhanced resistance toantibiotic therapy (28). CF is characterized by lung inflamma-tion mediated, in part, by chronic P. aeruginosa infection. P.aeruginosa possesses numerous virulence factors that facilitateevasion of the immune system (15, 37, 43, 49). For example, P.aeruginosa secretes enzymes such as alkaline protease and elas-tase, which degrade complement components and thus limitthe role of complement in the clearance of early pulmonary P.aeruginosa infections (16). The critical role of complement inthe clearance of P. aeruginosa is evidenced by the observationthat C3 and C5 knockout mice were unable to clear P. aerugi-nosa after challenge (40, 69). In addition, P. aeruginosa ex-

presses lipopolysaccharide variants that interfere with C3bdeposition (52).

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

P. aeruginosa possesses two types of flagellins, type A andtype B, that differ in amino acid composition and length of thehypervariable region. P. aeruginosa flagellins have the uniqueproperty of being potent adjuvants as well as protective anti-gens (8, 32, 42, 50). Previous work has established flagellin asa potent adjuvant in mice (1, 3, 9, 10, 23, 33–35, 45, 53, 56) aswell as cynomolgus and African green monkeys (24, 36). Aphase III clinical trial of P. aeruginosa flagellins in CF patientsdemonstrated that the vaccine was well tolerated and caused a30% reduction in the incidence of infection (12). In relatedstudies, immunization with the OprI antigen of P. aeruginosaand an appropriate adjuvant elicited a protective response inmice that correlated with the titer of OprI-specific immuno-globulin G (IgG) (14). In addition, an adenovirus expressingepitope 8 (amino acids 311 to 341) of OprF (i.e., the OprF311-341

protein) provided protection against acute P. aeruginosa infec-tion (67, 68). Several investigators have focused on a fusionpeptide containing OprF and OprI as a potential vaccine can-

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Wake Forest University School of Medicine,Medical Center Blvd., Winston-Salem, NC 27157. Phone: (336) 716-2216. Fax: (336) 716-9928. E-mail: [email protected].

� Published ahead of print on 6 April 2009.

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didate. Although large amounts of this protein were requiredfor an optimal response, immunization with an OprF-OprIfusion protein resulted in a 95-fold increase in the 50% lethaldose for mice. A subsequent study in burn patients revealedthat an OprF-OprI fusion protein was immunogenic and welltolerated (26, 31).

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

MATERIALS AND METHODS

Strains and plasmids. Bacterial strains and plasmids used in this study aredescribed in Table 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, 5g/liter yeast extract). Solid media were prepared by adding 1.0 to 1.5% Selectagar (Gibco-BRL). Plasmids in E. coli were selected using media supplementedwith antibiotics (carbenicillin, 100 �g ml�1; gentamicin, 10 �g ml�1). Plasmids inP. aeruginosa were selected on media containing carbenicillin (300 �g ml�1),gentamicin (100 �g ml�1), and Irgasan (25 �g ml�1). E. coli strain JM109 wasused for all cloning procedures, while E. coli SM10 was used to transferplasmids into P. aeruginosa by biparental mating (60). The P. aeruginosastrains used were PAO1 and its derivatives WFPA850, WFPA852, WFPA854,WFPA860, WFPA862, WFPA864, and WFPA866. Vectors pEX18Gm andpEX18Ap or derivatives were used for cloning and gene replacements (Table 1).

Construction of nonpolar fliC, oprF, and oprI deletion mutations. To engineerunmarked, nonpolar fliC, oprF, and oprI deletion mutations, we utilized a pre-viously described method (57). Internal fragments of coding sequences within

each gene were deleted using a modified PCR technique termed splicing byoverlap extension (65). In this assay, four gene-specific primers were employed inthree separate PCRs to generate DNA fragments with a defined in-frame dele-tion of coding sequences within the fliC, oprF, or oprI genes. The primers werealso designed such that the final amplicon, harboring the specified mutated alleleharbored restriction sites to allow direct cloning into pEX18Ap or pEX18Gm,resulting in plasmid pHL150 (�fliC), pHL153 (�oprF), or pHL155 (�oprI). Themutant alleles were introduced into the PAO1 chromosome as outlined previ-ously (21). The merodiploids were resolved by growing on sucrose-containingmedia and introduction of the mutated allele, which was verified by PCR.

Recombinant proteins. DNA encoding full-length type A flagellin of P. aerugi-nosa strain PAK and DNA encoding full-length type B flagellin of strain PAO1were each amplified by PCR and ligated into pET29a. DNA encoding the matureOprI antigen of P. aeruginosa strain PAO1 (amino acids 21 to 83) was amplifiedby PCR and ligated into pET29a or to the 5� end of type A and B flagellin genesin pET29a, generating constructs that encode OprI-type A flagellin and OprI-type B flagellin (OprI-flagellins). DNA encoding epitope 8 (amino acids 311 to341) of OprF of P. aeruginosa strain PAO1 was amplified by PCR and ligated intopET29a or to the 5� end of the OprI and type A flagellin gene construct and theOprI and type B flagellin gene construct. The structure of each final protein ispresented in diagrammatic form in Fig. 1.

All expressed proteins were purified by metal ion affinity chromatography aspreviously described (1, 24). Acrodisc Q membranes were used to deplete en-dotoxin and nucleic acids. Endotoxin levels were �10 pg/�g 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-� and antigen-specific IgG. Tumor necrosis factor alpha(TNF-�) 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 independentexperiments with triplicate samples in each experiment.

Titers of antigen-specific IgG were measured using MaxiSorp plates coatedwith 100 �l of antigen (type A flagellin, type B flagellin, OprI, or OprF) at 10�g/ml in sterile phosphate-buffered saline (PBS). The plates were incubatedovernight at 4°C and then blocked with 10% newborn calf serum in PBS. Plasmasamples (in triplicate) were added, and the plates were incubated overnight at4°C, followed by secondary anti-Ig antibodies (Roche Diagnostics) for 2 h atroom temperature. Peroxidase activity was detected with 3,3�,5,5�-tetramethyl-benzidine (TMB) liquid substrate system (Sigma-Aldrich) and stopped with 2 NH2SO4. Endpoint dilution titers were defined as the inverse of the lowest dilutionthat resulted in an absorbance value (at 450 nm) 0.1 higher than that of naiveplasma. Groups of at least seven mice were used. To determine relative antibodyaffinities, the ELISA was conducted as described above with the addition of a15-min incubation with sodium thiocyanate (NaSCN) (Sigma) solution as de-scribed previously (1, 29).

Mice. Six- to 8-week-old BALB/c and DBA/2 mice were purchased fromCharles River Laboratories. All animals were maintained under pathogen-freeconditions. All research performed on mice in this study complied with federaland institutional guidelines set forth by the Wake Forest University Animal Careand Use Committee.

Intramuscular immunization of mice. Groups of seven mice were anesthetizedwith 2,2,2-tribromoethanol (Avertin; Sigma) and tert-amyl alcohol (Fisher) byintraperitoneal injection. Small volumes (20 �l total) containing antigen andadjuvant in PBS were injected using a 29.5-gauge needle into the right calf ofeach mouse. Mice were boosted at 4 weeks via the same route and bled twoweeks later. Plasma was prepared and stored at �70°C until analysis.

ELISPOT assay. The frequency of antigen-specific plasma cells was deter-mined using limiting dilution analysis as previously described (54). Briefly, Im-mobulin-P high-affinity protein binding enzyme-linked immunospot (ELISPOT)

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

TABLE 1. Bacterial strains used in this study

Strain Description or characteristic(s)a Source orreference

PAK WT 59PAO1 WT 59WFPA850 In-frame fliC deletion in PAO1 This studyWFPA852 In-frame oprF deletion in PAO1 This studyWFPA854 In-frame oprI deletion in PAO1 This studyWFPA860 In-frame fliC and oprI deletions

in PAO1This study

WFPA862 In-frame fliC and oprF deletionsin PAO1

This study

WFPA864 In-frame fliC, oprF, and oprIdeletions in PAO1

This study

WFPA866 In-frame oprF and oprIdeletions in PAO1

This study

T69833 Mucoid CF isolate D. J. Wozniak,unpublished

1286 Nonmucoid CF isolate D. J. Wozniak,unpublished

PDO300M Mucoid PAO1 30PDO300NM Nonmucoid PD0300 deficient in

alginate production30

a WT, wild type.

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plates (Millipore) were coated with 100 �l of type A flagellin, type B flagellin,OprI, or OprF (10 �g/ml) in sterile PBS. Bone marrow (BM) and spleen werecollected 45 days postboost, single-cell suspensions were prepared, and dilutionsof the cells (5 � 105/well) were added to the antigen-coated wells. Plates werethen incubated at 37°C for 5 h, washed, and probed with goat anti-mouseantibody (4°C overnight). Plates were developed using horseradish peroxidase-Avidin D diluted 1:1,000 (Southern Biotechnology) and 3-amino-9-ethylcardba-zole (AEC) and dried overnight. Spots were enumerated using a dissectingmicroscope. Only wells that contained �4 spots were counted for analysis. Totalspleen plasma cell numbers were calculated by multiplying the number of cells inthe spleen by the number of spots per million spleen cells. Total BM plasma cellnumbers were calculated in the same manner with an additional multiplication by7.9 to compensate for total BM (2).

To determine the frequency of antigen-specific memory B cells (MBC), theBM and spleen cells were incubated in vitro for 5 days in the presence of 1 �g/mlOprF311-341–OprI–flagellins and then plated as described above. The number ofMBC was determined by subtracting the number of plasma cells from the 5-hincubation from the total number of plasma cells after the 5-day culture. Resultsfor two independent experiments are given.

Antigen-specific IgG binding to P. aeruginosa. P. aeruginosa strains were in-cubated with heat-inactivated control or immune mouse plasma for 1 h at 4°Cprior 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 samplesin each experiment.

Antigen-specific IgG-mediated complement activation. Control and immunemouse 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 nmof 0.5 (108 CFU/ml), washed two times with sterile PBS, and then incubatedwith mouse plasma for 1 h. The bacteria were then washed and incubated for 1 hat 37°C with 5% rabbit serum (Innovative Research) as a source of complement.Finally, the bacteria were stained with goat anti-rabbit C3-fluorescein isothio-cyanate (MP Biomedical). Flow cytometric analysis was performed using a BDFACSCalibur, and data were analyzed with FloJo software (Tree Star, Inc.,Ashland, OR). Histograms representative of results from three experiments areshown. Complement-mediated killing was performed as described above with theexception that the bacteria were incubated for 4 h with rabbit serum. A timecourse experiment revealed minimal killing at 1 h with rabbit serum (data notshown). The percentage of bacteria killed was quantitated by the followingequation: (number of input bacteria � number of recovered bacteria)/(numberof input bacteria) � 100.

Respiratory challenge with agar-embedded P. aeruginosa. P. aeruginosa strainswere grown in LB broth lacking NaCl to 108 CFU/ml. One part bacteria wasadded 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 min-eral 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 withsterile PBS. The final volume was adjusted to approximately 5 ml. To determinethe number of CFU/ml, the agar-bacterium beads were homogenized, and beadsize was determined by comparison to 100- to 150-�m chromatography beads.Mice (six or seven per group) were anesthetized with 2,2,2-tribromoethanol (withtert-amyl alcohol) by intraperitoneal injection, and then 50 �l of agar-embeddedbacteria was instilled intratracheally using a sterile gel-loading tip.

Histology. Lungs were harvested and transferred to 10% formalin for 24 h. Thetissue was then trimmed, embedded in paraffin, cut into 4-�m sections, and stainedwith hematoxylin and eosin by routine methods. For histological examination,groups of four mice were used for each condition. Slides were blindly scored on anincreasing severity index that incorporates values for consolidation, bronchiolar andvascular degenerative changes, and edema (range for each factor, 0 to 4). Totalinflammation score was calculated by the sum of all categories. Representativeimages (see Fig. 9) are shown from four animals/group, with three sections peranimal.

Statistical analyses. Statistical analysis was performed using SigmaStat 3.10 (Sys-tat Software, Inc., Point Richmond, CA). For normally distributed data sets, signif-icance was determined using Student’s t test. The significances of data sets whichwere not normally distributed or were of unequal variances were determined usingthe Mann-Whitney rank sum test. Where applicable, a two-way analysis of variancetest was applied. P values of less than 0.05 were considered significant.

RESULTS

TLR5-specific signaling activity of P. aeruginosa type A andB flagellins and OprF311-341–OprI–flagellins. In order to gen-

erate antigens with flagellin as the adjuvant, we generatedseveral constructs as shown in Fig. 1. In view of the insertion ofthe OprF and OprI sequences at the N terminus of flagellin, itwas important to determine if this addition would have a neg-ative impact on the ability of each flagellin, i.e., type A or B, tosignal via TLR5. To test the ability of P. aeruginosa flagellinseither alone or as part of a three-part fusion with OprF andOprI (Fig. 1) to signal via TLR5, RAW 424 (TLR5�) or RAW264.7 (TLR5�) cells were incubated with 1 pM to 1 nM of eachprotein, and production of TNF-� was assessed. Stimulation ofRAW 424 cells with P. aeruginosa type A or B flagellin, OprIand type A or B flagellin, or OprF311-341, OprI, and type A orB flagellin resulted in a concentration-dependent increase inTNF-� (Fig. 2A to C). In contrast, none of these proteinsinduced TNF-� production in cultures of TLR5� RAW 264.7cells. Consistent with previous results with the P. aeruginosaflagellins, the half-maximal stimulation occurred at 16 pM fortype A flagellin and 40 pM for type B flagellin (8). There wasno significant difference between the half-maximal stimula-

FIG. 2. TLR5-specific signaling activity of P. aeruginosa type A andB flagellins and OprF311-341–OprI–flagellin. RAW 424 (TLR5�) andRAW 264.7 (TLR5�) cells were stimulated with 10�9 to 10�12 M ofprotein. At 4 h poststimulation, supernatants were harvested and theamount of TNF-� was determined by ELISA. (A) Type A and Bflagellins. (B) OprI -flagellin fusions. (C) OprF311-341–OprI–flagellinfusions. Data represent the results of three independent experimentsdone in triplicate.

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tions of type A or B flagellin, OprI and type A or B flagellin,or OprF311-341, OprI, and type A or B flagellin. Thus, thepresence of OprF-OprI at the N terminus of type A or Bflagellin does not alter recognition and signaling via TLR5.

Immunization with OprF311-341–OprI–flagellins promotes apotent antigen-specific humoral response. To assess the abilityof OprF311-341–OprI–flagellins to promote an antigen-specifichumoral response, groups of seven BALB/c or DBA/2 micewere immunized with 5 �g of each flagellin (types A and B)plus 10 �g OprI, 5 �g OprI–flagellin fusion proteins, or 5 �gOprF311-341–OprI–flagellin fusion proteins. Prior experimentsestablished that immunization of BALB/c mice with 5 �g OprI-flagellins generated a maximal IgG response to flagellin andOprI (data not shown). Control mice received either OprI orOprF311-341–OprI at equivalent molar doses. DBA/2 mice wereused because previous studies identified DBA/2 mice as moresusceptible to P. aeruginosa infection than BALB/c andC57BL/6 mice (55, 58). Four weeks later, mice were boosted inan identical manner. Two weeks after the boost, the mice werebled and plasma was prepared for analysis of circulating anti-gen-specific IgG. Mice immunized with OprI-flagellins orOprF311-341–OprI–flagellins exhibited a robust OprI-specificIgG response (Fig. 3). In contrast, there was no significantOprI-specific IgG response in mice given only OprI or type Aand B flagellins with OprI. In all cases, flagellin-specificresponses were extremely robust. Mice immunized withOprF311-341–OprI–flagellins exhibited a high level of OprF-specific IgG as well as flagellin- and OprI-specific IgG.

In addition to determining the titers of antigen-specific IgGfollowing immunization with OprF311-341–OprI–flagellins, wealso evaluated IgG isotypes and IgE. Plasma was preparedfrom immune mice as described above, and antigen-specificIgG subclasses and IgE were determined by ELISA. Immuni-zation of mice with OprF311-341–OprI–flagellins did not elicitany detectable antigen-specific IgE (data not shown). Thisfinding is consistent with our prior work demonstrating thatflagellin 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 find-ing is consistent with our prior work on flagellin as an adjuvantin a Yersinia pestis vaccine (24).

Generation of antigen-specific plasma cells and MBC inresponse to OprF311-341–OprI–flagellins. In view of the robustantigen-specific IgG response, we evaluated the frequency ofantigen-specific plasma cells and MBC generated in responseto OprF311-341–OprI–flagellins. Mice were immunized with 5�g of OprF311-341–OprI–flagellins as described above, and 45days postboost, BM and spleens were harvested and the fre-quencies of antigen-specific plasma cells and MBC were de-termined by ELISPOT assay. Antigen-specific plasma cellswere determined following 5 h incubation. Eighty-five percentof antigen-specific plasma cells were found in the BM, and15% were found in the spleen. Consistent with the IgG titerdata (Fig. 3), there were more plasma cells for type A and Bflagellins (200/106 BM cells) than for OprI (42) and OprF(30) (Fig. 4A). No plasma cells were detected in wells thatcontained cells from nonimmune mice. Although significantlymore antigen-specific plasma cells were found in the BM, asubstantial number of plasma cells remained in the spleen (Fig.4B). The retention of antigen-specific plasma cells in thespleen correlated with the immunogenicity of each antigen.

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

OprF311-341–OprI–flagellin immunization generates high-affinity antigen-specific IgG. Since antigen affinity plays a crit-ical role in the functional activity of an antibody, we evaluatedthe relative affinity of the IgG generated following immuniza-tion with OprF311-341–OprI–flagellins. The relative affinity ofantibodies can be assessed in an ELISA by determining theconcentration of sodium thiocyanate required to reduce anti-body binding by 50%. As shown in Fig. 5, immunization withOprI-flagellins or OprF311-341–OprI–flagellins generated IgGwith equivalent relative affinities for the three antigens. Forcomparative purposes, a vaccine consisting of flagellin plus Y.pestis F1 antigen generated F1-specific IgG requiring 3 Msodium thiocyanate for 50% reduction in binding (1). Giventhe observation that these antibodies provide complete protec-tion against respiratory challenge with Y. pestis (24, 36), wedefined high-affinity IgG as antibodies requiring 2 to 3 Msodium thiocyanate for 50% reduction in antigen binding.OprF311-341–OprI–flagellin immune plasma had an averagerelative IgG affinity approaching 3 M for flagellins, OprI, andOprF (Fig. 5). Thus, the data are consistent with the conclu-sion that OprF311-341–OprI–flagellins elicits high-affinity anti-gen-specific IgG.

Complement-activating activity of antibodies specific forOprI, OprF, and type A and B flagellins. To assess the func-tional activity of each of the antigen-specific IgG types, it wasfirst necessary to generate P. aeruginosa mutants lacking one ormore of these antigens (Table 1; see also Materials and Meth-ods). The type B flagellin-expressing P. aeruginosa strain PAO1

FIG. 3. Immunization with OprF311-341–OprI–flagellin promotesa potent antigen-specific humoral response. BALB/c or DBA/2 micewere immunized intramuscularly with 5 �g of type A and B flagel-lins plus 10 �g OprI, 5 �g OprI–type A flagellin plus 5 �g OprI–typeB flagellin, or 5 �g OprF311-341–OprI–type A flagellin plus 5 �gOprF311-341–OprI–type B flagellin. At 4 weeks postimmunization,animals were boosted, and 2 weeks postboost, blood was collectedand antigen-specific total IgG was determined by ELISA. Datarepresent at least seven mice per group in triplicate. Asteriskscorrespond to differences compared to values for “A-flagellin�B-flagellin�OprI,” with a P value of �0.05 by a Mann-Whitney ranksum test.



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was used as the genetic background for the mutants. Eachmutant exhibited growth kinetics that were similar to that ofthe wild-type strain (data not shown). P. aeruginosa strainswere incubated with immune or control mouse plasma at 4°Cfor 1 h and then stained for the presence of IgG. As shown inFig. 6A, wild-type P. aeruginosa bound significant amounts of

IgG specific for flagellin, OprI, and OprF. Furthermore, mu-tants positive for only flagellin, OprF, or OprI also bound highlevels of IgG. Experiments using a type A flagellin-expressingstrain, PAK, demonstrated similar results (data not shown).These results demonstrate that the antibodies generatedagainst the recombinant fusion protein recognize these anti-gens in their cell-associated forms. This is particularly impor-tant in the case of OprF, since only a single epitope was presentin the OprF311-341–OprI–flagellin fusion proteins.

Having established the ability of the individual populationsof IgG to recognize the cell-associated antigens, we next eval-uated the potential of these antibodies to activate complement.Previous work has clearly established the importance of thecomplement system in the clearance of P. aeruginosa (13, 40,52, 69). To assess the ability of IgG antibodies specific forOprF, OprI, and type A and B flagellins to activate comple-ment, we measured the extent of IgG-dependent C3 depositionon P. aeruginosa. The various P. aeruginosa strains were incu-bated with a 1:10 dilution of heat-inactivated immune mouseplasma for 1 h, and then 5% rabbit complement was added foran additional hour. The bacteria were then stained with fluo-rescein isothiocyanate-labeled C3-specific antibody, and theextent of C3 deposition was determined by flow cytometry. Atime course revealed that 1-h incubation with serum was opti-mal for C3 deposition and yielded minimal cell death (data notshown). As a control, we used a P. aeruginosa strain lackingflagellin, OprI, and OprF. OprI-flagellin or OprF311-341–OprI–flagellin immune plasma promoted significant C3 depositionon the surface of wild-type P. aeruginosa (Fig. 6A). By usingmutants that lack one or more of the eliciting antigens, wefound that IgG with specificity for each of the eliciting antigenspromoted robust C3 deposition (Fig. 6B and C). When allthree antigens were present, there was a synergistic increase inthe level of C3 deposition. These results indicate that OprF311-341–OprI–flagellin immunization generated antigen-specific IgGthat exhibited a high degree of functional activity and that thecombination of flagellin-, OprI-, and OprF-specific IgG anti-bodies triggered the highest level of C3 deposition.

FIG. 4. Generation of antigen-specific plasma cells and MBC byOprF311-341–OprI–flagellin immunization. DBA/2 mice were immu-nized intramuscularly with 5 �g of OprF311-341–OprI–flagellins. BMand spleens were harvested 40 days postboost and analyzed for anti-gen-specific plasma and MBC by ELISPOT assay. (A) Frequencies ofantigen-specific plasma cells and MBC. (B) Total numbers of antigen-specific plasma cells and MBC. Results are the averages from twoindependent experiments on five mice.

FIG. 5. OprF311-341–OprI–flagellin immunization generates high-affinity antigen-specific IgG. Plasma samples from mice that receivedOprI-flagellins or OprF-OprI-flagellins were used to determine rela-tive antibody affinities for type A flagellin, type B flagellin, OprI, andOprF. Antigen-specific IgG affinity was determined by ELISA usingdilutions of sodium thiocyanate (NaSCN). Data are presented as molarconcentrations of NaSCN required to reduce absorbance 50%. Sam-ples are from the same mice that were used in the experiments whoseresults are presented in Fig. 2 and 3. There were seven mice per group,with each sample done in triplicate.



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Antibody-dependent complement-mediated killing of P.aeruginosa by OprF311-341–OprI–flagellin immune plasma. Inview of the robust ability of OprF-, OprI-, and type A and Bflagellin-specific IgG antibodies to promote C3 deposition, wenext examined the ability of those antibodies to promote com-plement-mediated killing of P. aeruginosa. Bacteria were incu-bated with heat-inactivated immune plasma for 1 h, and then5% 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 nonspe-cific killing by normal serum (data not shown). Approximately90% of wild-type, nonmucoid P. aeruginosa (PAO1, PAK, and1286) isolates as well as strains expressing type B flagellin,OprI, or OprF were susceptible to antibody-dependent com-plement-mediated killing (Fig. 7 and Table 2). In contrast, only18% of mucoid P. aeruginosa (T68933 and PDO300M) isolateswere susceptible to killing (Table 2). This result is not unex-pected, given the presence of a large amount of alginate ex-opolysaccharide in the mucoid strains that would likely maskOprI and OprF. In support of this conclusion, we found that astrain of PAO1 (PDO300NM) deficient in alginate production(and thus nonmucoid) was quite sensitive to killing (Table 2).In addition, the generally applicable inverse relationship be-tween flagella and alginate expression (59) would also limit theeffectiveness of the flagellin-specific IgG. The antigen depen-dence of the killing was evidenced by the very low level ofkilling, with bacteria lacking all three of the eliciting antigens.When the source of complement was heat inactivated, onlybackground levels of killing were observed. Taken together,these findings clearly demonstrate that the antibodies gener-ated in response to OprF311-341–OprI–flagellins exhibit potentantigen binding, complement-activating activity, and killing ofnonmucoid but not mucoid P. aeruginosa.

FIG. 6. Complement-activating activity of OprI, OprF, and type Aand B flagellin-specific IgG antibodies. Plasma samples from OprF311-341–OprI–flagellin-immunized mice were incubated with P. aeruginosa, andIgG binding and C3 deposition was determined by flow cytometry. Theantigens expressed by each P. aeruginosa strain are shown. (A) Antigen-specific IgG binding to P. aeruginosa. (Left column) Plasma samples fromOprI-flagellin-immunized mice. Filled regions refer to control strainWFPA860 (�fliC �oprI), which lacks flagellin and OprI. (Right column)Plasma samples from OprF-OprI-flagellin-immunized mice. WT, wildtype; B-Flagellin�, fliC� �oprI �oprF strain; OprI�, oprI� �fliC �oprFstrain; OprF�, oprF� �fliC �oprI strain. Filled regions refer to controlstrain WFPA864 (�fliC �oprI �oprF), lacking all three antigens. (B) C3deposition on P. aeruginosa strains. FITC, fluorescein isothiocyanate.(C) Percentages of C3-positive strains from the data shown in panel B. *,P value of �0.05; **, P value of �0.001 in a comparison with WFPA860(�fliC �oprI) (top) or WFPA864 (�fliC �oprI �oprF) (bottom). Statisticswere determined using Student’s t test. Data are from two independentexperiments performed in triplicate.

FIG. 7. Antibody-dependent complement (Comp)-mediated killingof P. aeruginosa by OprF311-341–OprI–flagellin-immunized mouseplasma. Plasma samples from OprF311-341–OprI–flagellin-immunizedmice were diluted 1:10 and heat inactivated at 56°C for 1 h. Sampleswere then supplemented with 5% rabbit complement for 4 h at 37°C.Data are from four samples over two experiments, each sample donein duplicate. *, P value of �0.05 by Student’s t test.

TABLE 2. Complement-mediated killing of additionalP. aeruginosa strains

Straina % Killed

PAK (WT) ..................................................................................86.5 0.21286 (nonmucoid CF isolate) ...................................................84.3 0.8PD0300 M (mucoid PAO1)......................................................15.2 1PD0300NM (alginate-deficient PDO300) ...............................83.4 0.4T68933 (mucoid CF isolate).....................................................24.8 0.3

a WT, wild type.



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Enhanced clearance of P. aeruginosa in OprF311-341–OprI–flagellin-immunized mice. Nonmucoid P. aeruginosa does notcause a chronic infection in healthy mice as it does in CFpatients. If large doses of bacteria are used, the mice quicklysuccumb to bacteremia (E. T. Weimer, D. J. Wozniak, andS. B. Mizel, unpublished observations). With small doses, themice rapidly clear the bacteria. In view of the lack of a suitableanimal model that closely mimics the situation in CF patients,i.e., chronic infection, investigators have evaluated agar-em-bedded mucoid P. aeruginosa as a way to infect mice such thatrapid septic shock is avoided and the time of infection islengthened (25, 58). For example, Stevenson and colleagues(55, 58) used the agar bead model to demonstrate that DBA/2mice were more susceptible to mucoid P. aeruginosa than wereBALB/c or C57BL/6 mice. However, since the initial P. aerugi-nosa infection in CF patients is mediated by nonmucoid strains(5, 28, 61), we felt it was more appropriate to use nonmucoidbacteria in the agar bead model. Preliminary results revealedthat unimmunized mice did not succumb when infected intra-tracheally with up to 3.5 � 106 CFU of nonmucoid P. aerugi-nosa embedded in agar beads, but the mice did exhibit sub-stantial morbidity. In view of the finding that DBA/2 mice aremore susceptible to P. aeruginosa, we used this strain to eval-uate the ability of OprF311-341–OprI–flagellin immunization topromote enhanced clearance of P. aeruginosa embedded inagar. DBA/2 mice were immunized as described above andinfected intratracheally with 3.5 � 106 CFU of agar-embeddedPAO1. Lungs were harvested 1, 3, and 5 days postinfection,and bacteria were enumerated by serial dilutions on LBNSplates. One day after challenge, immunized mice displayed amarked decrease in bacterial burden compared to controlmice (Fig. 8). After 3 days, five of six mice immunized withOprF311-341–OprI–flagellins had cleared the infection. In con-trast, the control mice had large numbers of bacteria in thelungs. Although the control mice cleared the infection by day5, our results clearly demonstrate that immunization withOprF311-341–OprI–flagellins had a dramatic effect on the rateof bacterial clearance. It is important to emphasize that theability of mice immunized with OprF-OprI to clear the infec-tion by day 5 reflects a limitation of this model and not theefficacy of the OprF311-341–OprI–flagellin vaccine.

Reduced lung pathology following pulmonary P. aeruginosachallenge in OprF311-341–OprI–flagellin-immunized mice. Inaddition to determining bacterial burden following challenge,we also evaluated the histopathology of lungs from mice im-munized with OprF311-341–OprI–flagellins or OprF-OprI.Lungs were harvested 1, 3, and 5 days after P. aeruginosachallenge. One day after P. aeruginosa challenge, alveolar wallsfrom OprF311-341–OprI–flagellin-immunized mice displayedslight thickening owing to congestion and increased numbersof inflammatory cells. In contrast, lungs from mice immunizedwith OprF-OprI developed bronchopneumonia, with airway-oriented neutrophils, edema, and abundant visible bacteria(Fig. 9A). After 3 days, immune mice exhibited only minorinflammatory changes in the lung, whereas more severe pneu-monia with diffuse consolidation was present in the controlanimals. After 5 days, the lungs of immune mice were normal,while those of the controls had thickened alveolar walls, aresult of congestion and inflammatory cells (Fig. 9B). In sum-mary, mice immunized with OprF311-341–OprI–flagellins dis-played minimal lung pathology which completely resolved byday 5 postchallenge. The absence of lung pathology in theimmune mice not only demonstrates the efficacy of the vaccinein promoting bacterial clearance but also the ability of thevaccine to promote clearance without inducing secondary tis-sue damage. In striking contrast, mice immunized with OprF-OprI demonstrated severe pneumonia which only partially re-solved by day 5 (Fig. 9A). In conjunction with the results of invitro experiments (Fig. 5 and 6), it is clear that OprF311-341–OprI–flagellin immunization promotes an adaptive immuneresponse that promotes the generation of antigen-specific IgGthat exhibits robust functional activity, facilitates rapid clear-ance, and prevents the development of severe pneumonia fol-lowing P. aeruginosa infection.

DISCUSSION

The goal of this study was twofold: to establish a set ofcriteria for a vaccine against P. aeruginosa and then to developand test the vaccine based on these criteria. Based on ourresults, we conclude that the OprF311-341–OprI–flagellin vac-cine meets all of the proposed criteria: the vaccine containsflagellin, a potent adjuvant, is multivalent, generates high-titerantigen-specific IgG that exhibits a high degree of functionalactivity, generates a robust memory response, and enhancesclearance of nonmucoid P. aeruginosa without secondary tissuedamage. Although the antigen-specific IgG induced by thisvaccine did not promote complement-mediated killing of mu-coid P. aeruginosa, it is important to emphasize that longitudi-nal studies of CF patients have clearly demonstrated that theinitial P. aeruginosa infection is mediated by nonmucoid bac-teria (5, 28, 61). We wish to emphasize that although theOprF311-341–OprI–flagellin vaccine has many of the features ofan efficacious vaccine, the evaluation of its potential for use inCF patients is limited at this time by the lack of a suitableanimal model that closely mirrors the situation in CF patients.

In addition to promoting high-level antigen-specific IgG(Fig. 3), flagellin also promoted the generation of significantnumbers of antigen-specific MBC (Fig. 4). In view of theseobservations, it is likely that the very limited reduction in theincidence of P. aeruginosa infections in CF patients immunized

FIG. 8. OprF311-341–OprI–flagellin-immunized mice display en-hanced rate of clearance following pulmonary P. aeruginosa challenge.DBA/2 mice were immunized twice with 5 �g of OprF311-341–OprI–flagel-lins and challenged intratracheally with 3.5 � 106 CFU of agar-embeddedPAO1. The right lungs were harvested 1, 3, and 5 days postinfection, andbacterial burden was assessed by counting. Data are the averages for six orseven mice per group. The dotted line indicates the limit of detection. #,P value of 0.053; **, P value of 0.002 by Mann-Whitney rank sum test.



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FIG. 9. OprF311-341–OprI–flagellin-immunized mice are protected against severe lung pathology during pulmonary P. aeruginosa chal-lenge. The left lungs of identical mice used the experiments represented in Fig. 7 were evaluated for histology. Lungs were fixed in 10%formalin for 24 h and paraffin embedded, and 4-�m sections were cut. Slides were stained with hematoxylin and eosin. Representative imagesare shown from three sections/animal. (A) OprF-OprI-immunized mice. Panels A1 to A3 are images magnified �4. Panel A4 is an imagemagnified �40 to show bacteria. Panels A5 and A6 are images magnified �20. (B) OprF311-341–OprI–flagellin-immunized mice. Panels B1to B3 are images magnified �4. Panels B4 to B6 are images magnified �20. (C) Slides were blindly scored for consolidation, bronchiolarand vascular degenerative changes, alveolar wall thickness, and edema. The score for inflammation was determined by the sum for eachcategory. Dotted line indicates lowest score possible. *, P value of �0.05 by Student’s t test.



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with sheared flagella (12) may be due to a low level of func-tional adjuvant activity of the material. Although the adjuvantsPam3Cys and Pam2Ser (19) as well as alum (63) were found toenhance the response to P. aeruginosa antigens, it appears thatflagellin is far more effective, as evidenced by the dramaticdifferences in the amounts of antigen required and the result-ant titers of antigen-specific IgG.

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

The titers of flagellin-, OprI-, and OprF-specific IgG anti-bodies following immunization with OprF311-341–OprI–flagel-lins are in most cases two logs higher than those reported inother studies (12, 14, 22, 50, 51, 67, 68, 70). For example, vonSpecht et al. (62) required three immunizations with 70-foldmore antigen to achieve equivalent antibody responses. Thedifference may be due to the extraordinary potency of flagellinas an adjuvant, as well as the use of fusion proteins that en-hance the efficiency of antigen delivery to dendritic cells via thebinding of the associated flagellin to TLR5 on these cells (1a).The finding that immunization with OprF311-341–OprI–flagel-lins promotes the generation of large numbers of plasma cellsis consistent with the very high titers of induced IgG. Further-more, the generation of immunologic memory is evidenced bythe 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 theclearance of P. aeruginosa in mice. In addition, we have dem-onstrated that antibodies specific for each antigen activatecomplement and mediate killing of P. aeruginosa (Fig. 6 and 7).In this regard, von Specht and colleagues (13) found that thelevel of complement-activating OprI-specific IgG correlatedwith the level of protection against P. aeruginosa. Our resultsclearly support this conclusion. The finding that intramus-cular immunization with OprF311-341–OprI–flagellins pro-motes clearance is consistent with the conclusion thatprotection is not dependent on the availability of antigen-specific IgA. In this regard, Pier et al. (44) demonstratedthat intraperitoneal immunization could prevent mucosalcolonization by P. aeruginosa.

A number of studies (4, 17, 38, 39) have presented evidencein support of the conclusion that heightened production of Th2cell-derived cytokines, such as interleukin-4 (IL-4), IL-10, andIL-13, is associated with a poor prognosis in CF patients. Inview of the notion that isotype switching to IgG1 is Th2 celldriven, the question arises as to whether the observed IgG1bias (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 patientsis based on cytokine production and not IgG isotype. Indeed,two studies revealed that elevated levels of alginate-specificIgG2 and IgG3, and not IgG1, are associated with a poorprognosis in CF patients (46, 47). It is also important to notethat class switching to IgE is also promoted by Th2 cells, yet wehave never observed any increase in IgE in response toOprF311-341–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 aclassic Th2 response, and thus, the possibility that a flagellin-based P. aeruginosa vaccine might cause substantial secondarytissue damage is not warranted. This conclusion is clearly sup-ported by the observation that the lungs of mice immunizedwith OprF311-341–OprI–flagellins did not exhibit any evidenceof residual tissue damage following clearance of the bacteria(Fig. 9).

Although OprF311-341–OprI–flagellins promotes more rapidclearance of bacteria in healthy mice, it remains to be deter-mined if it will be efficacious in CF patients. The pathophysi-ologic events that occur in CF patients as well as the virulencefactors expressed by P. aeruginosa (66) clearly represent asignificant challenge for any vaccine. The presence of a defec-tive CFTR leads to abnormal ion transport and water deple-tion in the airway of CF patients that ultimately results in anincrease in fluid viscosity, impaired mucociliary clearance, in-creased bacterial trapping in the mucus layer facilitatingchronic infection, and a reduction in antibody penetration (18).In addition, infections by S. aureus and H. influenzae and laterP. aeruginosa contribute to the development of a hyperinflam-matory environment in the respiratory tract (7). All of theseevents contribute to a vicious cycle of infection and inflamma-tion that ultimately causes severe pathology. Chronic inflam-mation can adversely affect vaccine efficacy due to the high-level production of prostaglandins that exert an inhibitoryeffect on lymphocytes (6). In addition, chronic inflammationpromotes the continual recruitment of neutrophils to the lungthat in turn release elastase that induces tissue damage andrelease of DNA, a substance that provides a scaffold for P.aeruginosa biofilms (20, 64).

The major cause of chronic inflammation in CF patients ispersistent P. aeruginosa infection. Overproduction of alginateand, subsequently, biofilm development allow P. aeruginosa toevade the immune system and also increase antibiotic resis-tance, making it extremely difficult to eradicate the infection(5, 28). Mucoid conversion is also associated with a significantdecrease in lung function in CF patients (41). Our findingsindicate the best time to vaccinate CF patients using theOprF311-341–OprI–flagellin fusion would be prior to mucoidconversion, since complement-mediated killing occurred onlyin nonmucoid bacteria (Fig. 7 and Table 2). Thus, the patho-physiologic events associated with CF in combination with thepathogenic mechanisms associated with P. aeruginosa repre-sent a significant challenge to the elements of protective im-munity. It is our view that vaccine efficacy will be most effectiveat a very early stage in the disease process. Perhaps the limitedsuccess of previous vaccine studies was not solely due to thelimited inherent efficacy of the vaccines but rather to the tim-ing of immunization.

ACKNOWLEDGMENTS

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 theCystic Fibrosis Foundation (MIZEL0810) and NIH grant AI061396(to D.J.W.).

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A Fusion Protein Vaccine Containing OprF Epitope 8, OprI, and Type