Dual-Function Vaccine for Pseudomonas aeruginosa ...

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INFECTION AND IMMUNITY, 0019-9567/01/$04.000 DOI: 10.1128/IAI.69.11.6962–6969.2001 Nov. 2001, p. 6962–6969 Vol. 69, No. 11 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Dual-Function Vaccine for Pseudomonas aeruginosa: Characterization of Chimeric Exotoxin A-Pilin Protein RALF HERTLE, 1 † RANDALL MRSNY, 2 AND DAVID J. FITZGERALD 1 * Biotherapy Section, Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, Maryland 20892-4255, 1 and Genentech, Inc., South San Francisco, California 94080-4990 2 Received 26 June 2001/Returned for modification 26 July 2001 /Accepted 7 August 2001 Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we char- acterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutra- lize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseu- domonas colonization in susceptible individuals. Colonization of cystic fibrosis (CF) individuals with Pseudo- monas aeruginosa represents a significant negative milestone in the progression of this disease. Once colonized, patients are subject to the damaging effects of various secreted virulence factors and to the inflammatory response of the host immune system. A key component of colonization is the adhesion of type IV pili to asialo-GM1 receptors on the surface of epithe- lial cells (26, 45, 48; for a review, see reference 21). Type IV pili are composed of pilin polymers arranged in a helical structure with five subunits per turn (19, 41). The portion of the pilin protein responsible for cell binding is located near the C ter- minus (amino acids 129 to 142) in a -turn–-turn loop sub- tended from a disulfide bond (5, 6, 23, 36). A 12- or 17-amino- acid sequence (depending on the specific strain) in this loop interacts with receptors on epithelial cells. For CF individuals, the overproduction of the R domain of the mutant CF trans- membrane conductance regulator can lead to an increased level of asialo-GM1 and, accordingly, an increased binding of P. aeruginosa (3, 26, 45). Functional studies of pilin have indi- cated that only the last pilin subunit (the tip) of a pilus inter- acts with epithelial cell receptors (31). To interfere with bac- terial adhesion, anti-pilin antibodies will need to recognize residues that are normally located at the C-terminal loop of pilin (32). Structural studies have indicated that this loop is dominated by main chain residues; and this may explain why pilins from distinct strains bind the same receptor despite se- quence variation and the presence of both 12- and 17-amino- acid loops. Generating antibodies to the C-terminal pilin loop may be useful in reducing or eliminating colonization (15, 22, 47). Table 1 lists the pilin loop sequences from several strains of P. aeruginosa. Pseudomonas exotoxin A (here called PE), a prominent vir- ulence factor secreted by P. aeruginosa, is cytotoxic for mam- malian cells by virtue of its ability to enter cells by receptor- mediated endocytosis and then, after a series of intracellular processing steps, translocate to the cell cytosol and ADP-ribo- sylate elongation factor 2 (17, 25, 38, 39). This results in the inhibition of protein synthesis and cell death. It is possible to generate a nontoxic mutant toxin that has no ADP-ribosylating activity PE (reference 33 and this study). PE is composed of three prominent structural domains and one minor subdomain (Fig. 1) (1). The N-terminal domain (Ia) is responsible for receptor binding and the middle domain (II) has translocating activity, while the C-terminal domain (III) is an ADP-ribosyl transferase (24). Subdomain Ib (located between domains II and III in the primary sequence) has no known function and can be deleted without loss of toxin activ- ity. As a virulence factor, PE can kill polymorphonuclear leuko- cytes, macrophages, and other elements of the immune system (44). In this way, toxin-mediated destruction of local immune cells may contribute to the maintenance of P. aeruginosa in- fections (43, 52). The importance of PE as a virulence factor has been confirmed by results showing that toxin-producing strains are more virulent than nontoxogenic ones (53) and by data from murine models of Pseudomonas infection where the presence of anti-PE antibodies reduced pathogenicity and ex- tended life (16, 42, 49). Here, we report on the development with a wholly recom- binant vaccine. The deletion of glutamic acid at position 553 of PE (PE553) produces a protein that exhibits all toxin func- tions with the exception of ADP-ribosylation (33). PE553, which is noncytotoxic for cells, animals, or humans, is a poten- tial platform for vaccine development. Between domains II and III is the small subdomain termed Ib. It is composed of a seven-amino-acid loop subtended from a disulfide bond. Be- cause deletion of this structure to produce a protein we term PE64 (Fig. 1) causes no loss of toxin activity, it is an attractive * Corresponding author. Mailing address: Biotherapy Section, Lab- oratory of Molecular Biology, CCR, National Cancer Institute, Bldg. 37, 4B03, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-9457. Fax: (301) 402-1969. E-mail: [email protected]. † Present address: Institut fuer Mikrobiologie, D-72076 Tuebingen, Germany. 6962 on April 14, 2018 by guest http://iai.asm.org/ Downloaded from

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INFECTION AND IMMUNITY,0019-9567/01/$04.00�0 DOI: 10.1128/IAI.69.11.6962–6969.2001

Nov. 2001, p. 6962–6969 Vol. 69, No. 11

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Dual-Function Vaccine for Pseudomonas aeruginosa: Characterizationof Chimeric Exotoxin A-Pilin Protein

RALF HERTLE,1† RANDALL MRSNY,2 AND DAVID J. FITZGERALD1*

Biotherapy Section, Laboratory of Molecular Biology, CCR, National Cancer Institute, Bethesda, Maryland20892-4255,1 and Genentech, Inc., South San Francisco, California 94080-49902

Received 26 June 2001/Returned for modification 26 July 2001 /Accepted 7 August 2001

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies toprevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we char-acterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutra-lize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were insertedinto a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termedPE64�553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterizedby various biochemical and immunological assays. PE64�553pil bound specifically to asialo-GM1, and, wheninjected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killingactivity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseu-domonas colonization in susceptible individuals.

Colonization of cystic fibrosis (CF) individuals with Pseudo-monas aeruginosa represents a significant negative milestone inthe progression of this disease. Once colonized, patients aresubject to the damaging effects of various secreted virulencefactors and to the inflammatory response of the host immunesystem. A key component of colonization is the adhesion oftype IV pili to asialo-GM1 receptors on the surface of epithe-lial cells (26, 45, 48; for a review, see reference 21). Type IV piliare composed of pilin polymers arranged in a helical structurewith five subunits per turn (19, 41). The portion of the pilinprotein responsible for cell binding is located near the C ter-minus (amino acids 129 to 142) in a �-turn–�-turn loop sub-tended from a disulfide bond (5, 6, 23, 36). A 12- or 17-amino-acid sequence (depending on the specific strain) in this loopinteracts with receptors on epithelial cells. For CF individuals,the overproduction of the R domain of the mutant CF trans-membrane conductance regulator can lead to an increasedlevel of asialo-GM1 and, accordingly, an increased binding ofP. aeruginosa (3, 26, 45). Functional studies of pilin have indi-cated that only the last pilin subunit (the tip) of a pilus inter-acts with epithelial cell receptors (31). To interfere with bac-terial adhesion, anti-pilin antibodies will need to recognizeresidues that are normally located at the C-terminal loop ofpilin (32). Structural studies have indicated that this loop isdominated by main chain residues; and this may explain whypilins from distinct strains bind the same receptor despite se-quence variation and the presence of both 12- and 17-amino-acid loops. Generating antibodies to the C-terminal pilin loopmay be useful in reducing or eliminating colonization (15, 22,47). Table 1 lists the pilin loop sequences from several strainsof P. aeruginosa.

Pseudomonas exotoxin A (here called PE), a prominent vir-ulence factor secreted by P. aeruginosa, is cytotoxic for mam-malian cells by virtue of its ability to enter cells by receptor-mediated endocytosis and then, after a series of intracellularprocessing steps, translocate to the cell cytosol and ADP-ribo-sylate elongation factor 2 (17, 25, 38, 39). This results in theinhibition of protein synthesis and cell death. It is possible togenerate a nontoxic mutant toxin that has no ADP-ribosylatingactivity PE (reference 33 and this study).

PE is composed of three prominent structural domains andone minor subdomain (Fig. 1) (1). The N-terminal domain (Ia)is responsible for receptor binding and the middle domain (II)has translocating activity, while the C-terminal domain (III)is an ADP-ribosyl transferase (24). Subdomain Ib (locatedbetween domains II and III in the primary sequence) has noknown function and can be deleted without loss of toxin activ-ity.

As a virulence factor, PE can kill polymorphonuclear leuko-cytes, macrophages, and other elements of the immune system(44). In this way, toxin-mediated destruction of local immunecells may contribute to the maintenance of P. aeruginosa in-fections (43, 52). The importance of PE as a virulence factorhas been confirmed by results showing that toxin-producingstrains are more virulent than nontoxogenic ones (53) and bydata from murine models of Pseudomonas infection where thepresence of anti-PE antibodies reduced pathogenicity and ex-tended life (16, 42, 49).

Here, we report on the development with a wholly recom-binant vaccine. The deletion of glutamic acid at position 553 ofPE (PE�553) produces a protein that exhibits all toxin func-tions with the exception of ADP-ribosylation (33). PE�553,which is noncytotoxic for cells, animals, or humans, is a poten-tial platform for vaccine development. Between domains IIand III is the small subdomain termed Ib. It is composed of aseven-amino-acid loop subtended from a disulfide bond. Be-cause deletion of this structure to produce a protein we termPE64 (Fig. 1) causes no loss of toxin activity, it is an attractive

* Corresponding author. Mailing address: Biotherapy Section, Lab-oratory of Molecular Biology, CCR, National Cancer Institute, Bldg.37, 4B03, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255. Phone:(301) 496-9457. Fax: (301) 402-1969. E-mail: [email protected].

† Present address: Institut fuer Mikrobiologie, D-72076 Tuebingen,Germany.

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location for the insertion of third-party sequences, especiallyloop sequences. Previously, we reported that the Ib loop couldbe replaced by sequences from the V3 loop of HIV gp120 (18).Inserts of 14 or 26 amino acids were accommodated withoutdisturbing PE functions (18).

To produce a chimeric protein that displays pilin in a near-native conformation, we replaced the Ib domain of PE byamino acids 129 to 142 of pilin (Fig. 1) including the disulfidebond that links cysteines 129 to 142. This chimeric protein ischaracterized here as a candidate vaccine designed to produceantibodies that will interfere with Pseudomonas adherence andneutralize PE.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strains, plasmids, andoligonucleotides used in this study are listed in Table 2. Pseudomonas strainsused for adherence studies were grown on Luria-Bertani agar and then in M9minimal medium (KD Medical, Bethesda, Md.) supplemented with 0.4% glucose

at 30°C without shaking. Cultures in late log phase were routinely used foradhesion assays.

Oligoduplex formation and plasmid construction. A 54-bp sense oligonucle-otide with cohesive ends for PstI and encoding the 12-amino-acid pilin loop ofthe PAK strain was annealed with a 54-bp antisense oligonucleotide in 10 mMTris-HCl and 50 mM NaCl (pH 7.4) (oligonucleotide sequences are listed inTable 2). Annealing was accomplished by heating to 94°C for 5 min followed bycooling to 25°C over a period of 40 min. Plasmids pPE64 and pPE64�553 (seeTable 2), encoding enzymatically active and inactive PE, respectively, were di-gested with PstI at residue 1470 (see FitzGerald et al. [18]). Ligation with thephosphorylated pilin oligoduplex destroyed the PstI site and introduced a uniqueSpeI site. A XhoI/SpeI double digest was used to check for the correct orientationof the insert. Final constructs were verified by dideoxy double-strand sequencing.

Antibodies and proteins. The PK99H mouse monoclonal antibody and puri-fied pilin protein were gifts from Randall Irvin, University of Alberta, Alberta,Canada. Horseradish peroxidase-conjugated anti-mouse immunoglobulin G(IgG) and anti-rabbit IgG antibodies (Jackson ImmunoResearch Laboratories,West Grove, Pa.) were used at a 1:2,000 dilution to detect primary antibodies inWestern blots and enzyme-linked immunosorbent assays (ELISAs).

Chimera protein expression and purification. Chimeric proteins were ex-pressed in Escherichia coli and recovered from inclusion bodies as previouslydescribed (4). Briefly, strain BL21(�DE3) was transformed with plasmids har-boring a T7 promoter upstream of the initial ATG of the toxin-expressingvectors. Cultures were grown in Superbroth (KD Medical) with ampicillin(50 �g/ml) and then induced for protein expression by the addition of IPTG(isopropyl-D-thiogalactopyranoside) (1 mM). After 2 h of further culture, bac-terial cells were harvested by centrifugation. Following cell lysis, expressed pro-teins were recovered in inclusion bodies. Proteins were solubilized with guani-dine HCl (6.0 M) and 2 mM EDTA (pH 8.0) plus dithioerythreitol (65 mM).Solubilized proteins were then refolded by dilution into a redox-shuffling buffer(4). Refolded proteins were dialyzed against 20 mM Tris and 100 mM urea (pH7.4); adsorbed on Q Sepharose (Amersham Pharmacia Biotech); washed with150 mM NaCl, 20 mM Tris, and 1 mM EDTA (pH 6.5); and eluted with 280 mMNaCl, 20 mM Tris, and 1 mM EDTA. Eluted proteins were diluted fivefold and

TABLE 1. P. aeruginosa pilin loop sequencesa

Strain Sequenceb

Short pilin loopPAK CTSDQ-----DEQFIPKGCT2A CTSTQ-----DEMFIPKGCPAO, 90063 CKSTQ-----DPMFTPKGCCD, PA103 CTSTQ-----EEMFIPKGCK122-4 CTSNA-----DNKYLPKTCKB7, 82932, 82935 CATTV-----DAKFRPNGC1071* (GenBank no., AF331069) CESTQ-----DPMFTPKGC

Long pilin loop577B CNITKTPTAWKPNYAPANC1244, 9D2, P1 CKITKTPTAWKPNYAPANCSBI-N* (GenBank no. AF331072) CGITGSPTNWKANYAPANC

a Sequences are from database searches and from direct sequencing of pilingenes (this study). *, loop sequences that do not appear in current databases arereported here for the first time.

b Short pilin loop strain sequences are from cysteine 129 to cysteine 142; longpilin loop strain sequences are from cysteine 133 to cysteine 151.

FIG. 1. Shown in cartoon form is the domain organization of PE(1). PE64 lacks the loop region of domain Ib. PE64pil includes theinsertion of the pilin loop (residues 129 to 142) of the PAK strain ofP. aeruginosa. The deletion of glutamic acid 553 (indicated by a dot)removes an active site residue (33) and produces proteins PE64�553and PE64�553pil with no ADP-ribosylating activity. The Ib loop isshown in light shading and the pilin loop in darker shading.

TABLE 2. Strains, plasmids, peptides, and oligonucleotides

Name or sequence

StrainsP. aeruginosa

PAKPAO1SBI-Na

1071a

M2a

82935a

82932a

90063a

E. coli BL21(�DE3)

PlasmidspPE64pPE64�553pPE64pilpPE64�553pil

PeptidesKCTSDQDEQFIPKGCSKDEQFIPKQIDPEFK

Oligonucleotides (pilin loop duplex)Sense 5�-TTGTACTAGTGATCAGGATGAACAGTTTATTCCGAAAGGTTGTTCACGTATGCA-3�

Antisense 5�-TACGTGAACAACCTTTCGGAATAAACTGTTCATCCTGATCACTAGTACAATGCA-3�

a These strains were provided by I. A. Holder (Hospital for Sick Children,Cincinnati, Ohio).

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then adsorbed onto a MonoQ column (HR 10/10; Amersham Pharmacia Bio-tech) and further purified by the application of a linear salt gradient (0 to 0.4 MNaCl in Tris-EDTA, pH 7.4). PE proteins eluted between 0.2 and 0.25 M NaCl.Final purification was achieved with a gel filtration column (Superdex 200;Amersham Pharmacia Biotech) in phosphate-buffered saline (PBS), pH 7.4.

Cell cultures. A549 (ATCC CCL-185), and L929 (ATCC CCL-1) cells weremaintained in Dulbecco’s modified Eagle’s medium F12 (DMEM F12) supple-mented with 10% fetal bovine serum, 2.5 mM glutamine, a standard concentra-tion of penicillin and streptomycin (100 U of penicillin/ml and 100 �g of strepto-mycin/ml; Gibco BRL, Grand Island, N.Y.) (further designated complete medium)in 5% CO2 at 37°C. Cells were fed every 2 to 3 days and passaged every 5 to 7 days.For assays, cells were seeded into 24- or 96-well plates and grown to confluence.

Quantification of bacterial adherence. To quantify the association of Pseudo-monas with A549 cells, we followed the adhesion assay described by Chi et al. (8).Briefly, A549 cells were grown in a 24-well plate (antibiotic-free medium) to adensity of approximately 2 � 104 cells per well. Cells were washed three times inHanks’ balanced salt solution without serum and were overlaid with 0.5 ml ofDMEM F12 complete medium without fetal bovine serum. A multiplicity ofinfection of 20 was achieved by adding 10 �l of an appropriate bacterial dilution.Plates were incubated for 1 or 2 h at 37°C and 5% CO2.

To remove unbound bacteria, cells were gently washed three times withHanks’ balanced salt solution. Cells were then fixed for 1 h in 3.7% paraformal-dehyde and 200 mM HEPES, pH 7.2. Cells were washed twice with saline andstained with 10% Giemsa stain for 10 min. Samples were washed three times withwater and examined under light microscopy at 400� magnification. Adherentbacteria were quantified by counting the number of cell-associated bacteria per100 A549 cells.

Determination of binding to asialo-GM1 by ELISA. Plates (96-well) werecoated with asialo-GM1 or monsialo-GM1 (Sigma Chemical Co., St Louis, Mo.)that had been solubilized in methanol. A 100-�l solution of ganglioside (5 �g/ml)was added to each well and evaporated at 4°C until dry. Wells were washed threetimes with PBS and blocked with fish gelatin-PBS (BioFX, Randallstown, Md.)for 16 h at 4°C. Test proteins in blocking buffer were added at various concen-trations. After incubation for 1 h at 22°C, the supernatant was removed andbound protein was detected with heat-inactivated anti-PE64�553pil serum (1:100) as the primary antibody. For competition studies, proteins at 0.2 �g/ml wereincubated with 2 �g of asialo-GM1 or monosialo-GM1/ml for 30 min at roomtemperature. Samples were then added to asialo-GM1-coated plates as above.

Cytotoxicity assay. The inhibition of protein synthesis by PE64 and PE64pil onL929 cells was determined as described previously (38). For assessing toxinneutralization activity, the same proteins (at 1 �g/ml) were incubated for 30 minat 22°C with rabbit sera diluted to 1:100. Samples at the appropriate dilutionwere added to individual wells containing L929 cells.

Production of polyclonal antibodies. PE64�553pil (200 �g per injection) wasinjected subcutaneously into a total of four rabbits: two rabbits were coinjectedwith Freund’s adjuvant while two rabbits received no adjuvant. Subsequentinjections included three biweekly injections at the same dose with or withoutincomplete Freund’s adjuvant. About 12 ml of serum was recovered biweeklyfrom each rabbit. The sera were heat inactivated for 20 min at 56°C, and dilutionsthereof were used for assays without further purification.

Syntheticpeptides.Peptide1(acetyl-KCTSDQDEQFIPKGCSK-NH2)contain-ing the complete C-terminal loop of PAK pilin protein, peptide 2 (acetyl-DEQFIPK-NH2) containing the core cell-binding sequence, and peptide 3 (acetyl-QIDPEFK-NH2) with the scrambled amino acids of peptide 2 were customsynthesized by Sigma Genosys. Peptide 1 was oxidized to allow the formation ofa disulfide bond. The same peptides were also synthesized with a biotin label.

Inhibition of adhesion. To assess antibody-mediated inhibition of adherence,anti-PE64�553pil rabbit sera were incubated at dilutions from 1:20 to 1:100 with4 � 105 bacteria at 22°C for 30 min. Bacteria were then centrifuged, resuspendedin DMEM without supplements, and added to confluent monolayers of A549cells at a multiplicity of infection of 20 for 1 to 2 h. Adherence was determinedas described above. Immune sera taken after the fourth injection were comparedto prebleed samples taken from the same rabbits.

RESULTS

Vaccine design. To generate a PE-based pilin vaccine, wesynthesized an oligonucleotide duplex that encoded amino ac-ids 129 to 142 of pilin from the PAK strain of P. aeruginosa.The construction of a nontoxic PE vector whereby a uniquePstI site was introduced in place of subdomain Ib was previ-

ously reported (18). Ligation of the pilin oligoduplex into thePstI-cut vector was followed by several characterization stepsto confirm the presence of the pilin insert in the correct ori-entation. The pilin oligoduplex was ligated into PE vectors toproduce the plasmids pPE64pil and pPE64�553pil (enzymati-cally active and inactive, respectively). Final constructs wereconfirmed by double-stranded DNA sequencing. Vectors wereconstructed without a bacterial secretion sequence, allowingrecombinant proteins to be expressed as inclusion bodies.

Protein expression and purification. Using the T7 expres-sion system described by Studier et al. (51), four PE-relatedproteins were expressed in E. coli. These were PE64, PE64�553,PE64pil, and PE64�553pil (Fig. 1). Each protein was ex-pressed separately and purified to near homogeneity. Expres-sion was induced by the addition of IPTG for 2 h, followed byharvesting of bacterial pellets. Inclusion bodies were recoveredfrom lysed bacteria. Proteins were then denatured and rena-tured from inclusion bodies as outlined in Materials and Meth-ods. Briefly, proteins were solubilized in guanidine HCl and areducing agent and then renatured with a redox-shufflingbuffer (4). Refolded proteins were dialyzed against Tris-urea,loaded onto Q Sepharose, and then recovered with a stepgradient (0.15 and 0.28 M NaCl). The proteins eluted at 0.28 MNaCl were diluted and applied to a MonoQ column which wasthen developed with a linear salt gradient. Gel filtration wasused as the final purification step for PE64�553pil.

Characterization of PE64pil proteins. Proteins were initiallyanalyzed by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (Fig. 2A and C). Substantially pure proteins wereisolated by using the purification scheme outlined above. ByWestern blot analysis, PE64pil and PE64�553pil proteins re-acted with PK99H, a monoclonal antibody to the C-terminalloop of pilin (Fig. 2B). The same antibody also reacted withsoluble preparations of these proteins, indicating that the pilininsert was exposed on the surface of the chimeric protein (datanot shown). PE proteins without inserts did not react with thePK99H antibody (Fig. 2B).

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(A and C) and Western blot analysis (B) of PE proteins and pilin. (A)Lanes 1 to 4 show substantially pure PE proteins (4 to 5 �g of proteinwas loaded per lane) after MonoQ chromatography. A small amount ofdimer is noted at 130 kDa. From left to right, the proteins loaded werePE64, PE64pil, PE64�553, and PE64�553pil. Purified PAK pilin wasadded to lane 5. (B) Lanes 6 to 10 contain the same proteins as shownin panel A but were probed with a monoclonal antibody to the pilin loop.(C) Lane 11 is PE64�553pil after gel filtration chromatography. Stan-dard proteins and their molecular masses in kilodaltons are indicated.

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To investigate the influence of the pilin insert on toxin struc-ture and function, the two enzymatically active proteins, PE64and PE64pil, were compared by a cytotoxicity assay. Concen-trations of PE64 or PE64pil ranging from 0.002 to 20 ng/mlwere added to L929 cells for an overnight incubation. Activitywas then determined by measuring the inhibition of cellularprotein synthesis. Results indicated that PE64 and PE64pilexhibited similar toxicities with 50% inhibitory concentrationvalues in the range of 0.1 ng/ml for both proteins (Fig. 3). Thisresult suggested that the insert of the 12-amino-acid pilin se-quence did not unduly perturb toxin function and, by infer-ence, toxin structure.

To test the functionality of the pilin insert in the PE64proteins, we assayed various concentrations of PE64pil forreactivity with immobilized asialo-GM1. Previous results indi-cated that synthetic peptides derived from the C terminus ofpilin could bind asialo-GM1 and thereby block the binding ofpili to epithelial cells (27, 54). Increasing concentrations ofPE64pil from 0.1 to 2.0 �g/ml reacted specifically with immo-bilized asialo-GM1 (Fig. 4A). PE64 was used as a control andexhibited only a low level of binding (Fig. 4A). Additionalstudies were carried out to confirm the ganglioside specific-ity of both PE64pil and PE64�553pil. Soluble asialo-GM1 re-duced the binding of PE64pil and PE64�553pil to immobilizedasialo-GM1 while the addition of monosialo-GM1 did not (Fig.4B and C). Neither ganglioside interfered with the low-levelbinding of PE64 and PE64�553 (Fig. 4B and C). Taken to-gether, these results not only confirmed the presence of reac-tive pilin sequences but revealed a gain of function for thePE64pil proteins.

Rabbit immune response to PE64�553pil. To test the abilityof the toxin-pilin protein to generate relevant antibody re-sponses, four rabbits were injected with the PE64�553pil pro-tein. Two rabbits (numbered 87 and 88) received the proteinplus adjuvant (complete Freunds for the first injection fol-lowed by incomplete Freunds for subsequent injections), andtwo rabbits (numbered 89 and 90) received the protein alone.Two hundred micrograms of protein per injection was givensubcutaneously for a total of four cycles spaced approximately

2 weeks apart (Fig. 5). Anti-pilin titers were determined usingan ELISA assay where biotinylated pilin peptides were immo-bilized on strepavidin-coated plates. Over the period of immu-nization, anti-pilin titers increased in all four animals (Fig. 5).However, the speed and extent of the response were greater inthe two rabbits that received antigen plus adjuvant. To avoidcomplement-mediated bacterial killing (see below), immunesera were heat inactivated. This treatment did not significantlyalter antibody titers in the ELISA assay (data not shown).

Inhibition of P. aeruginosa (PAK strain) adhesion by postimmunization sera. Sera taken 2 weeks after the last injectionwere assayed for blocking activity by the bacterial adherenceassay. Compared to prebleeds, immune sera at various dilu-tions blocked adherence of the PAK strain of P. aeruginosa(Fig. 6A). Reduction of adherence ranged from 60% at adilution of 1:100 to 90% at a dilution of 1:20. At a dilution of1:20, blocking activity was comparable without regard to thepresence of adjuvant in the antigen preparation (Fig. 6B).

Inhibition of P. aeruginosa (various strains) Adhesion bypostimmunization sera. Inhibition of PAK strain adhesionconfirmed that rabbits responded to the specific pilin sequencethat was administered in the vaccine. However, because theC-terminal loop of pilin exhibits considerable sequence varia-tion, it was important to determine the reactivity of the im-mune sera for other strains of P. aeruginosa. Strains PAO1,1071, SBI-N, 82935, 82932, 90063, 1244, and M2 were testedfor adherence to A549 cells under conditions similar to thoseused for the PAK strain. The specific cell binding of all strainswas reduced in adhesion when heat-inactivated immune rabbitsera were mixed with bacteria at a 1:20 dilution (Fig. 6C). Thereduction in adhesion among the different strains was more orless in the range of that for the PAK strain (about 90% reduc-tion).

While it was unlikely that each of the above strains expressedthe same loop sequence as the PAK strain, it was of interest toanalyze variations at this portion of the pilin gene. Pilin se-quences were determined by generating PCR clones of eachstrain’s pilin gene and sequencing these. Primers for amplifi-cation were from the 5� end of the pilin gene and the 3� end ofthe neighboring gene (nicotinate-nucleotide pyrophosphory-lase) in the Pseudomonas genome (unpublished data). Resultsrevealed the following: most strains exhibited a 12-amino-acidloop while one, SBI-N, had a 17-amino-acid loop. Strains82932 and 82935 had the same loop sequence as KB7(SwissProt accession no. Q53391) and 90063 had a loop thatmatched PAO1 (PIR accession no. A25023). Strains 1071 andSBI-N exhibited loops with novel sequences (see Table 1).Strain M2, a mouse isolate, was not sequenced.

Toxin-neutralizing response. We also evaluated rabbit anti-sera for toxin-neutralizing activity. All four of the immunizedrabbits receiving a 1:20 dilution of sera neutralized 1.0 �g oftoxin/ml completely (Fig. 7). From these results we concludedthat the PE-pilin vaccine can generate antibodies of two reac-tivities: one that blocks adhesion and one that neutralizes theexotoxin.

DISCUSSION

Within the first year of life, 25% of CF individuals arecolonized with P. aeruginosa; by age 15, this percentage climbs

FIG. 3. Toxicity of PE64pil compared to PE64. To assess the effectof introducing a third-party loop into PE, we compared the toxicity ofPE64 (f) with PE64pil (Œ). Increasing concentrations of each proteinwere added to L929 cells and, after an overnight incubation, inhibitionof protein synthesis was determined. Results are expressed as percentcontrol compared to cells receiving no toxin. Error bars represent 1standard deviation (SD) of the mean from triplicate wells.

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toward 100% (2). Clearly, strategies to prevent the initial col-onization event are needed (2). Here, we report on the devel-opment of a chimeric subunit vaccine for generating antibodiesthat interfere with two important components of Pseudomonasvirulence, namely pilin-mediated adherence and the tissue de-structive activity of PE. Twelve amino acids from the C-termi-nal loop of pilin (PAK strain) were inserted at a location innontoxic PE where they could fold into a near-native confor-mation and cause little or no disruption of toxin structure. Pilinfunctionality was confirmed by showing that the chimeric pro-tein acquired the ability to bind asialo-GM1. Previously, it wasreported that the V3 loop from gp120 of HIV1 could be ac-commodated in the same location, while retaining antibodyreactivity for conformational-dependent epitopes (18). The re-sult with the pilin insert confirms the broad utility of thistoxin-based system for insertion of third party sequences, es-pecially loop structures.

We injected PE64�553pil subcutaneously into rabbits asproof of the principle that antibodies with the desired speci-ficities could be produced in an animal. In the future, otherroutes of administration will be pursued, especially mucosaldelivery to airway epithelia. Previously, we compared the sub-cutaneous route with mucosal delivery of toxin-V3 loop pro-teins (37). Results of mucosal vaccination indicated that arobust anti-V3 loop response could be achieved with high titerresponses of both serum IgG and secretory IgA antibodies.

Because the toxin-pilin chimeric protein is a candidate vaccineto prevent Pseudomonas colonization in CF, it will be impor-tant to optimize vaccine delivery for mucosal antibody re-sponses at airway epithelia.

Type IV pili, which are composed of pilin homopolymers,are thought to be responsible for the initial binding event thatmediates adherence of several gram-negative pathogens tomammalian cells. For Pseudomonas pili, this interaction in-

FIG. 4. PE64pil and PE64�553pil interact with immobilized asialo-GM1. (A) Various concentrations of PE64pil or PE64 were added toplates coated with asialo-GM1, and binding was determined by reac-tivity with rabbit anti-PE followed by a peroxidase-labeled goat anti-rabbit IgG antibody. Absorbance at 450 nm was used to monitorbinding. (B and C) To investigate ganglioside specificity, a competi-tion assay was devised whereby soluble asialo-GM1 (aGM1) or mono-sialo-GM1 (GM1) at 2 �g/ml was preincubated with PE64pil (B) orPE64�553pil (C), and the percent residual binding determined asdescribed in the legend to panel A. For panels B and C, graphs showthe mean of a representative triplicate experiment. Error bars repre-sent 1 SD. N.A., no addition of competitor.

FIG. 5. Anti-pilin antibody titers (1:100) postimmunization withPE64�553pil with and without adjuvant. Sera were collected from eachof four rabbits (numbered 87 to 90) at various times, diluted 1:100, andthen added to strepavidin-coated plates that had been loaded withbiotinylated pilin peptides. Rabbit IgG was detected by the addition ofa peroxidase-conjugated goat anti-rabbit antibody. Rabbits 87 (F) and88 (E) received adjuvant while rabbits 89 (�) and 90 (ƒ) did not.

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volves the binding of the C-terminal loop of the last pilinsubunit to asialo-GM1 on the surface of epithelia (21). Pilin isa 144-amino-acid protein with its cell-binding loop locatedbetween amino acids 129 to 142. Apparently, only antibodies tothis loop interfere with adhesion. And while the middle portionof pilin is immunogenic, the C-terminal loop usually fails togenerate a strong antibody response (22). To overcome poorimmunogenicity, strategies to include strong adjuvants alongwith pilin sequences have been proposed (22). Here, we usedan active site deletion mutant of PE as a combination proteincarrier and protein adjuvant. This strategy has resulted in adual neutralizing response to both pilin and PE. In our vaccineprotein, we retain the toxin’s binding domain (Fig. 1) and thuspromote delivery of the pilin loop to cells expressing the toxinreceptor, the low-density lipoprotein receptor-related proteindesignated LRP (also known as CD91) (29). Because LRP iswidely distributed on cells and tissues, including macrophagesand other antigen presenting cells (30), we speculate that thePE-carrier system has certain attractive features. It was re-ported recently that the administration of PE to tracheal epi-

FIG. 6. Antibody-mediated interference with Pseudomonas adhesion to A549 cells. (A) The PAK strain of P. aeruginosa was incubated with 1:20to 1:100 dilutions of prebleed or immune (taken after the fourth injection of antigen) sera from rabbit 87. Bacteria were then added to cells, andthe percent adhesion was determined by comparison with bacteria that had been incubated in media alone. (B) A 1:20 dilution of sera from eachrabbit, prebleed and immune, was tested for antibody mediated interference. (C) Various strains of P. aeruginosa were incubated with immune sera(1:20) from one of the rabbits that received antigen alone (rabbit 90) and one that received antigen plus adjuvant (rabbit 87). For each panel, barsrepresent the number of bacteria per cell determined by examining 100 A549 cells. The error bars represent 1 SD from the mean of threeindependent experiments.

FIG. 7. Antibody-mediated neutralization of PE toxicity. Immunesera (Œ) or prebleed sera (f) were diluted 1:20 and mixed with PE64at 1.0 �g/ml. Samples were then diluted to the concentration indicatedand added to L929 cells for an overnight incubation. Results areexpressed as percent control of protein synthesis compared to cellsreceiving no toxin. Error bars represent 1 SD of the mean from trip-licate wells.

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thelia resulted in efficient toxin delivery to submucosal lymphnodes and spleen (13). This bodes well for the mucosal deliveryof a PE-based vaccine to CF airways.

The potential value of a Pseudomonas vaccine relates in partto its ability to protect individuals broadly from the strains thatare present in the environment. Based on the length of thepilin loop insert, there are two groupings for P. aeruginosa: onegroup with a 12-amino-acid sequence and one with a 17-amino-acid insert. Both loops apparently bind asialo-GM1 and arethought to exhibit similar structures. Reflecting this, we notethat our vaccine protein, containing a 12-amino-acid loop fromthe PAK strain, was able to generate antibodies that werereactive not only for strains with the shorter loop but also forthe SBI-N strain, which displayed the longer loop. Our studieshave also provided additional sequence data for pilin and pilinloop sequences. We report here two pilin loop sequences thathave not previously been entered in databases (Table 1). De-tails of complete pilin sequences will be presented in greaterdetail elsewhere.

Chronic pulmonary colonization by P. aeruginosa is associ-ated with a decline in the clinical course of CF patients. Fre-quently, antibiotic therapy, even via pulmonary delivery, failsto eradicate P. aeruginosa infections in these patients (50).Controlling P. aeruginosa infections, or better yet, preventingthem, has thus become a critical unmet medical need in thecare of CF patients (2). To address this, a number of vaccineapproaches have been explored, many focused on outer mem-brane constituents (35, 40, 46), some focused on toxins (7, 14,20, 34) and some focused on a combination approach (7, 9–12,28).

Here, we characterized a recombinant fusion protein as acandidate vaccine for generating anti-pilin and anti-toxin re-sponses that interfere with bacterial adhesion and neutralizeexotoxin A activity. Results obtained to date support furtherdevelopment and evaluation of this approach.

ACKNOWLEDGMENTS

We thank Randy Irvin for his kind gift of pilin protein and thePK99H monoclonal antibody. We are indebted to Alan Holder forsupplying strains of Pseudomonas and for his advice and encourage-ment.

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Editor: J. T. Barbieri

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