Construction and Characterization of a Live, Attenuated aroA ...0.5% glucose [42]) and MSM...

INFECTION AND IMMUNITY, Mar. 2002, p. 1507–1517 Vol. 70, No. 30019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.3.1507–1517.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction and Characterization of a Live, Attenuated aroA DeletionMutant of Pseudomonas aeruginosa as a Candidate Intranasal Vaccine

Gregory P. Priebe,1* Mary M. Brinig,2 Kazue Hatano,1 Martha Grout,1 Fadie T. Coleman,1Gerald B. Pier,1 and Joanna B. Goldberg2

Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School,Boston, Massachusetts,1 and Health Sciences Center, University of Virginia, Charlottesville, Virginia2

Received 26 July 2001/Returned for modification 22 October 2001/Accepted 12 December 2001

Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, butprotective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protectiveO-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P.aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarkedaroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutantof PAO1 is designated PAO1�aroA. The aroA deletion was confirmed by both PCR and failure of the mutantto grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity inmice, PAO1�aroA could be applied either intranasally or intraperitoneally at doses up to 5 � 109 CFU permouse without adverse effects. No dissemination of PAO1�aroA to blood, liver, or spleen was detected afterintranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of miceand rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterialantigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potentphagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediatedphagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live,attenuated P. aeruginosa strain PAO1�aroA appears to be safe for potential use as an intranasal vaccine andelicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.

Lipopolysaccharide (LPS)-smooth strains of Pseudomonasaeruginosa are significant pathogens for immunocompromised(11) and critically ill (52) patients and are the initial colonizingstrains in children with cystic fibrosis (3). In the setting ofnosocomial pneumonia, P. aeruginosa has a strikingly high at-tributable mortality (51). The prevalence and importance of P.aeruginosa in human infections, coupled with the increasinglyfrequent presence of antibiotic resistance in clinical isolates(10, 16), have generated a pressing need for an effective vaccine.

For many years, it has been clear that high-level immunity toP. aeruginosa infections can be mediated by antibodies to theLPS O antigen (also known as O side chain) (13). However,protective epitopes have proven to be poorly immunogenic,while nonprotective or minimally protective O-antigen epi-topes often elicit the best immune responses. P. aeruginosa iscurrently classified into 20 serogroups based on LPS O-antigendeterminants, with most serogroups possessing subtype strainshaving subtle variations in the O antigen. Thus, there are over30 subtypes based on LPS O-antigen chemical structure (27).Although O-antigen-based vaccines can elicit antibodies thatare protective in animal models, this protection is generallyseen only when the strains used to isolate the vaccine antigenare also used in the challenge studies (4, 5, 47, 48). Broad-based protection against other strains, even subtypes within thesame serogroup, is not reliably generated (18, 19). With theseobservations in mind, an O-antigen-based vaccine would needto be more than 20-valent (probably more than 30-valent, due

to additional subtypes). However, previous efforts to makeeven a divalent vaccine have been unsuccessful. When relatedO antigens (in the form of purified high-molecular-weight O-polysaccharide) from subtype strains within serogroup O2/O5were combined, the immune response to each individual com-ponent was diminished (19). Furthermore, Cryz and colleagueshave shown that an octavalent O-antigen-toxin A conjugatevaccine engendered opsonic antibody responses only againststrains used to manufacture the vaccine and did not protecthumans at risk for nosocomial P. aeruginosa infections afterpassive transfer of immunoglobulin G (IgG) isolated from vac-cinated individuals (5, 6, 8).

The inability to harness the protective efficacy of LPS O-antigen-elicited antibodies into an effective, broadly protectivevaccine suggests an important role for cellular immunity in thecontrol of P. aeruginosa infections, as does recent evidence (14,21, 46) that P. aeruginosa readily enters lung and corneal epi-thelial cells during infection. This cellular invasion is mediatedby interactions with the cystic fibrosis transmembrane conduc-tance regulator (44, 45, 60) and appears to result in apoptosisof infected cells (12, 20). We hypothesized that live, attenuatedP. aeruginosa vaccine strains could exploit this intracellularphase in the pathogenesis of P. aeruginosa infections and elicita broadly protective immune response.

We used a gene replacement system based on the Flp re-combinase to construct an unmarked aroA deletion mutant ofthe P. aeruginosa serogroup O2/O5 strain PAO1. This live,attenuated strain was used to immunize mice and rabbits viathe intranasal (i.n.) route, and the antisera were assessed byenzyme-linked immunosorbent assay (ELISA) and for opsonickilling activity. We have chosen to focus on i.n. immunization

* Corresponding author. Mailing address: Channing Laboratory,181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4248. Fax:(617) 525-2510. E-mail: [email protected].

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because it has been shown to be highly effective with a widevariety of pathogens in stimulating both local and systemicimmunity (17, 59) as well as immunity at distant mucosal sites(26). The results show that intranasal immunization of miceand rabbits with this live, attenuated P. aeruginosa vaccineelicits opsonic antibodies against multiple strains of the P.aeruginosa serogroup O2/O5 and, in rabbits, against severalserogroup-heterologous strains as well, indicating the signifi-cant potential of using such live, attenuated strains for vacci-nation against LPS-smooth strains of P. aeruginosa.

MATERIALS AND METHODS

Bacterial strains. The bacterial strains and plasmids used in these experi-ments, along with their phenotypes and sources, are listed in Table 1.

Construction of aroA deletion mutants. The aroA gene of P. aeruginosa PAO1was recognized to be present from the Pseudomonas Genome Project (http://www.pseudomonas.com). As depicted in Fig. 1, primers aroA-F2 (5�-GCACGACATCTTTCTCGCC-3�) and aroA-R2 (5�-CCTTCGAGAATGATGCGC-3�)were used to amplify DNA from PAO1 by PCR. The resultant 1.7-kb PCRproduct was ligated into plasmid pUCP18, which was then used to construct anintegration-excision vector based on the gene replacement system of Hoang et al.and Schweizer (23, 55), which initially results in a P. aeruginosa strain having adeletion for the aroA gene with an inserted gentamicin resistance marker (aacC1,the gentamicin acetyltransferase 3-1 gene) flanked by the Flp recombinase target(FRT) sequences. Briefly, the plasmid pMB11 was generated by insertional

inactivation of the aroA gene with the FRT-aacC1-FRT cassette followedby ligation into the gene replacement vector pEX100T (55) (a gift of H. P.Schweizer). The pEX100T vector carries a sacB sucrose sensitivity gene, conferscarbenicillin resistance, and requires a recombination event to be replicated.Plasmid pMB11 was transferred to P. aeruginosa PAO1 from Escherichia coliS17-1 (57) by biparental mating. Recipient cells were plated on cetrimide agarcontaining aromatic amino acids (50 �g/ml) with gentamicin (250 �g/ml) toselect for the acquisition of gentamicin resistance encoded by aacC1. Coloniesappearing on this medium were then replated on the same medium containingcarbenicillin (300 �g/ml) to confirm that the entire plasmid was inserted into thechromosome. Surviving colonies were then plated on L agar containing aromaticamino acids, gentamicin, and 5% sucrose, which induces loss of the pEX100T-encoded sacB gene. Colonies that grew on this medium were tested for carben-icillin sensitivity to ensure loss of the plasmid and were assessed for auxotrophyfor aromatic amino acids by using MSM (a minimal salts medium containing0.5% glucose [42]) and MSM supplemented with aromatic amino acids. Theresultant strain, PAO1�aroA-Gm, was confirmed by PCR with primers aroA-F3(5�-CCTGATTTATCTGGCCCAGC-3�) and aroA-R3 (5�-GCGCTCAACTTGTGCCCGG-3�).

To remove the gentamicin resistance marker and delete the aroA gene, plas-mid pFLP2, which contains the Flp recombinase, was transferred from E. coliSM10 to PAO1�aroA-Gm. Colonies were grown on MSM containing aromaticamino acids and carbenicillin (500 �g/ml) to select for pFLP2 (23) in P. aerugi-nosa. Resultant colonies were screened for gentamicin sensitivity (to confirm thedeletion of the gentamicin resistance cassette); the gentamicin-sensitive colonieswere then plated onto MSM containing aromatic amino acids and 5% sucrose toselect for loss of the pFLP2 plasmid. Loss of the plasmid and deletion of aroAwere confirmed by PCR and sequencing and by failure to grow on MSM in the

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Descriptiona Reference or source

P. aeruginosaPAO1 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2d M. VasilPAO1�aroA aroA deletion mutant of PAO1, LPS smooth, serogroup O2/O5, subtype epitopes O2a,O2d This studyPAO1�aroA (pUCP18) PAO1�aroA containing the empty vector pUCP18, CbR This studyPAO1�aroA (pMB1) PAO1�aroA containing the intact aroA gene on plasmid pMB1, CbR This studyAK44 LPS-defective derivative of PAO1 (absent O antigen, complete outer core) 30AK1012 LPS-defective derivative of PAO1 (absent O antigen, incomplete core) 25PAC1R Wild-type strain, LPS smooth, serogroup O3 53PAC557 LPS-rough derivative of PAC1R (absent O antigen, complete outer core) 53170003 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2b 19IATS O16 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2b, O2e 19Fisher IT-3 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2c 19170006 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2d, O2e 19170007 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitopes O2a, O2d, O2f 19Fisher IT-7 Wild-type strain, LPS smooth, serogroup O2/O5, subtype epitope O2a 19Fisher IT-4 Clinical isolate (bacteremia) designated Rhodes, LPS smooth, serogroup O1 50170001 Wild-type strain, LPS smooth, serogroup O3, subtype epitopes O3a, O3b 27, 28IATS O3 Wild-type strain, LPS smooth, serogroup O3, subtype epitopes O3a, O3b, O3c 27, 286294 Clinical isolate (corneal infection), LPS smooth, noncytotoxic, serogroup O6 BPEIb; 15, 506073 Clinical isolate (corneal infection), LPS smooth, cytotoxic, serogroup O11 BPEI; 15, 506077 Clinical isolate (corneal infection), LPS smooth, cytotoxic, serogroup O11 BPEI; 15, 506206 Clinical isolate (corneal infection), LPS smooth, cytotoxic, serogroup O11 BPEI; 15, 50

E. coliHB101 supE44 hsdS20(r�

Bm�B) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1, 2

S17-1 thi pro hsdR recA RP4-2-Tc::Mu Km::Tn7 57SM10 thi-1, thr leu tonA lacY supE recA::RP4-2-Tc::Mu 7

PlasmidspMB1 pUCP18-based plasmid containing the intact aroA gene, ApR This studypUCP18 Broad host range shuttle vector, ApR 55pMB4 Derivative of pMB1 containing the aroA gene disrupted by the aacC1 cassette from

pPS856, ApR/GmRThis study

pEX100T oriT� sacB� gene-replacement vector 23, 56pMB11 pEX 100T-based derivative of pMB4 containing the sacB sucrose-sensitivity gene,

CbR/GmR/sucSThis study; 23

pPS856 Plasmid containing the aacC1 gentamicin-resistance cassette flanked by theFlp-recombinase target sequences, ApR/GmR

23

pFLP2 Plasmid containing the Flp recombinase, ApR 23

a Abbreviations: Ap, ampicillin; Gm, gentamicin; Cb, carbenicillin; Km, kanamycin; suc, sucrose; Tc, tetracycline; superscript R, resistant; superscript S, sensitive.b BPEI, Bascom-Palmer Eye Institute, Miami, Fla.

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absence of aromatic amino acids. The resultant unmarked deletion mutant ofPAO1 is referred to as PAO1�aroA. Auxotrophism due to deletion of aroA wasconfirmed by transferring in either a control plasmid (pUCP18) or a plasmid(pMB1) containing the intact aroA gene by electroporation.

Preparation of bacterial inocula. Frozen bacterial stocks were plated andgrown overnight on tryptic soy agar at 37°C. For immunization, bacteria weresuspended in either normal saline or phosphate-buffered saline (PBS). Concen-trations were adjusted spectrophotometrically and confirmed by viable countsafter serial dilution and plating on tryptic soy agar. For i.n. immunization studiesusing heat-killed bacteria, the inoculum was prepared in PBS as described above,heated at 60°C for 1 h, cooled, resuspended by vortexing, and then used for i.n.application without washing.

Immunization of mice and rabbits. Six- to 8-week-old female C3H/HeN orBALB/c mice (Harlan Sprague-Dawley Farms, Chicago, Ill.) were housed undervirus-free conditions. Before immunization, mice were first anesthetized with 0.2ml of a mixture of ketamine (6.7 mg/ml) and xylazine (1.3 mg/ml) in 0.9% salineinjected intraperitoneally (i.p.). Immunization consisted of placing 10 �l of thebacterial inoculum on each nare (total, 20 �l per mouse). Escalating doses of 1 �108, 5 � 108, and 109 CFU were administered at weekly intervals. New ZealandWhite rabbits (Millbrook Breeding Labs, Amherst, Mass.) were immunized on asimilar schedule, followed by repeated intranasal boosting with doses of 109 CFUevery 2 to 4 weeks, all using inocula of 100 �l (50 �l per nare). Rabbits wereanesthetized with 2 ml of a mixture of atropine (0.4 mg), ketamine (80 mg), andxylazine (10 mg) injected subcutaneously prior to each immunization. Mice andrabbits were immunized with E. coli HB101 as a control, using identical schedulesand doses. All animal experiments complied with institutional and federal guide-lines regarding the use of animals in research.

Determination of internalized bacteria in infected lungs. Using methods pre-viously described (1), adult BALB/c mice were sacrificed at various times afteri.n. inoculation. After removal of the lungs in a sterile fashion, single-cell sus-pensions were obtained by passage through wire screens. Total bacterial numberswere determined by lysis of cells in Triton X-100 followed by serial dilution andplating. The numbers of internalized bacteria were determined by incubation ofthe lung suspensions in gentamicin at 37°C for 1 h followed by washing, lysingwith Triton X-100, diluting, and plating.

Histological analysis of lungs after i.n. application. Adult C3H/HeN micewere sacrificed at 0.5, 1.5, 3, 6, 24, and 48 h after i.n. inoculation with eitherPAO1 or PAO1�aroA. The lungs were immediately instilled with 1 ml of PBScontaining 1% paraformaldehyde by means of a catheter placed directly into thetrachea after exposure with a midline neck incision. The lungs were removed,fixed in PBS with 1% paraformaldehyde for 1 h at room temperature, and thenplaced in 70% ethanol in water at 4°C overnight prior to paraffin embedding.Sections were stained with hematoxylin and eosin.

ELISA and opsonophagocytic assays. ELISAs were performed by standardmethods as described previously (18). To assess heat-stable antigens, bacteria

FIG. 1. Construction of unmarked P. aeruginosa PAO1 with deletion of aroA. See Table 1 for a description of plasmids. Ap, ampicillin; Gm,gentamicin; Cb, carbenicillin; suc, sucrose.

FIG. 2. Total (A) and intracellular (B) bacteria in lungs of BALB/cmice at the indicated times following i.n. inoculation of 2 � 109 CFUof PAO1�aroA. Each time point represents data from five mice; errorbars indicate standard errors of the means.

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were heated at 95°C for 45 min in PBS, followed by centrifugation for 30 min at15,000 � g in a microcentrifuge. The pellet was resuspended in 0.01 M sodiumphosphate buffer (pH 7.0) and used to coat microtiter plates for ELISA. Op-sonophagocytic assays also used published methods (43), with the only differ-ences being that infant rabbit serum (Accurate Chemical, Westbury, N.Y.) wasused as the complement source and was not adsorbed with any organisms. Inaddition, bacteria were grown in tryptic soy broth containing 1% glycerol as asupplemental carbon source. Negative controls were antisera from mice or rab-bits immunized i.n. with E. coli HB101. Tubes with PAO1�aroA antisera butwithout polymorphonuclear leukocytes (PMNs) served as additional negativecontrols to help distinguish killing from agglutination. The positive control an-tisera were 1:4 dilutions of sera from rabbits immunized intravenously withheat-killed whole bacteria of the homologous serogroup (rabbit antiserum to theheat-killed Fisher IT-7 strain was the positive control for serogroup O2/O5strains). Antisera were adsorbed by incubating antisera with lyophilized bacteria(5 mg/ml) for 1 h at 4°C, removing the bacteria by centrifugation at 15,000 � g,and then filtering the supernatant through a 0.2-�m-pore-size filter. Antiserawere each adsorbed twice by the above procedure. For all assays, mouse serawere collected and pooled (four to five C3H/HeN mice per immunization group)3 weeks after the third immunization. Rabbit sera were collected 1 week after theseventh immunization.

Statistical analyses. ELISA titers were calculated by linear regression analysis ofduplicate or triplicate measurements of adjusted optical density values (with opticaldensity of normal mouse or rabbit sera subtracted) versus the log10 of the serum

dilutions. The x-intercept defined the endpoint titer. Calculation of 95% confidenceintervals for titers was done using the formula VT � (T2VB � 2 TCAB � VA)/B2,where T is the titer (log10); VT is the variance of the titer; VB is the variance of theslope, B, of the regression analysis; CAB is the covariance of the estimated interceptand slope; and VA is the variance of the intercept coefficient, A. VA, VB, and CAB

were obtained from the variance-covariance matrix of parameter estimates usingSPSS statistical software (SPSS, Chicago, Ill.). The 95% confidence intervals werethen calculated as T � 1.96(VT)1/2. The significance of the percentage of organismskilled in the opsonophagocytic assay was determined by analysis of variance(ANOVA) with Fisher’s protected least significant difference (PLSD) using Statview(SAS Institute, Cary, N.C.) with comparison to the E. coli HB101 control antisera.Under routine conditions, killing of 50% is considered biologically significant andtherefore serves to classify a serum as positive for opsonic killing activity. Thus,although killing of 50% is sometimes statistically significant, this level of killing isnot considered biologically significant.

RESULTS

Construction of the aroA deletion mutant. Using the genereplacement system of Hoang et al. and Schweizer (23, 55), weconstructed an unmarked aroA deletion mutant of the P.aeruginosa serogroup O2/O5 strain PAO1 (Fig. 1). The aroA

FIG. 3. Low-power views of hematoxylin-and-eosin-stained sections of lungs removed at the indicated time points from C3H/HeN mice infectedi.n. with PAO1 (left column) or PAO1�aroA (right column).



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deletion was confirmed by PCR and sequencing, and auxotro-phy for aromatic amino acids was verified by the inability togrow on MSM unless supplemented with aromatic amino acids(data not shown). Introduction of a plasmid containing the

intact aroA gene conferred the ability to grow on MSM withoutamino acids (data not shown).

Safety and biologic disposition in animals. We have re-ported that i.n. application of P. aeruginosa on the nares of

FIG. 4. IgG titers of antisera from C3H/HeN mice against P. aeruginosa serogroup O2/O5 (A) and heterologous (B) strains by ELISA. TheInternational Antigenic Typing Scheme (IATS) (31, 32) serogroup of each heterologous strain is indicated below the strain designation. Barsindicate endpoint titers as determined by linear regression, and error bars indicate the 95% confidence intervals.

FIG. 5. IgG titers of rabbit antisera against P. aeruginosa serogroup O2/O5 (A) and heterologous (B) strains by ELISA. The InternationalAntigenic Typing Scheme (IATS) (31, 32) serogroup of each heterologous strain is indicated below the strain designation. Bars indicate endpointtiters as determined by linear regression, and error bars indicate the 95% confidence intervals.

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anesthetized mice reliably produces rapid translocation to thelungs and is an excellent model for pneumonia and systemicspread (1). When doses of the attenuated strain PAO1�aroAup to 5 � 109 CFU were administered i.n. (or up to 1 � 109

CFU i.p.) to adult BALB/c or C3H/HeN mice, there were notoxic effects. In comparison, the 50% lethal dose for PAO1given i.n. is 3 � 107 CFU (1). Up to 109 CFU of PAO1�aroAgiven i.n. to anesthetized adult rabbits also had no apparenttoxicities. As shown in Fig. 2A, when PAO1�aroA was inocu-lated i.n. at 2 � 109 CFU per mouse, about 107 CFU per gramof lung was recovered immediately postinfection (in contrast tothe 67% of parental PAO1 cells that rapidly translocated to thelungs after i.n. infection [1]). The loss in viability of more than99.9% of the inoculum is presumably due to the rapid death ofthe aroA deletion mutant in the absence of aromatic aminoacids. Approximately 2 � 107 CFU of PAO1�aroA per gram oflung tissue was also recovered 4 h after infection, after whichthe number of recoverable CFU/gram of lung tissue droppedprogressively over time. By day 4 and after, all lungs weresterile. Interestingly, as depicted in Fig. 2B, the percentage ofthe surviving inoculum internalized by mouse lung cells, asdetermined by gentamicin exclusion assays, was essentially100% by 18 h after inoculation, probably due to the failureof the aroA deletion mutant to survive extracellularly inthe absence of aromatic amino acids. No dissemination ofPAO1�aroA to blood, liver, or spleen was detected (data notshown). Histological analysis of lungs from mice after i.n. in-oculation with PAO1 or PAO1�aroA (Fig. 3) showed similardegrees of mild inflammation at early time points (0.5 to 6 h,with representative sections at 6 h shown). However, by 48 hafter inoculation, the lungs of mice given PAO1 had evidenceof severe pneumonia with extensive PMN infiltration, alveolarhemorrhage, and filling of alveoli with proteinaceous debrisand bacteria. On the other hand, at 48 h, the lungs of micegiven PAO1�aroA had only mild to moderate inflammation,with overall preservation of alveolar and airway architecture.

Antibody responses after i.n. immunization. All immuniza-tions were done without adjuvants. When adult C3H/HeNmice were immunized i.n., high titers of serum IgG to wholebacterial cells were measured for serum collected 3 weeks afterthe last vaccination (Fig. 4). Titers to the parental strain PAO1were highest (20,000) and were cross-reactive with most se-rogroup-homologous strains (10,000 for five of the sevenprototypic serogroup O2/O5 strains; Fig. 4A) and several se-rogroup-heterologous strains (Fig. 4B). Repeated i.n. immuni-zation of rabbits with PAO1�aroA was also well tolerated and,after seven doses, elicited high titers of IgG (20,000) to wholebacterial cells of all seven of the prototypic serogroup O2/O5strains and to several serogroup-heterologous strains (Fig. 5).The antisera also possessed high IgG titers against heat-stablebacterial antigens (Fig. 4 and 5), which are predominantlycomposed of LPS epitopes (titers were 5,000 for heat-stableantigens of all seven prototypic serogroup O2/O5 strains forthe mouse antisera and 10,000 for six of the seven strainsusing the rabbit antisera).

The antisera mediated phagocytic killing of �50% of cells ofthe parental strain PAO1 at serum dilutions as high as 1:2,048for the mouse antiserum and 1:1,024 for the rabbit antiserum(Fig. 6). Remarkably, when compared with antiserum from arabbit immunized intravenously with heat-killed (60°C for 1 h)

Fisher IT-7, the antisera from these i.n.-immunized animalshad only two- to fourfold-lower activity in the opsonophago-cytic assays. Using serum dilutions of 1:8, five of the sevenprototypic serogroup O2/O5 strains were killed (50% killing)by the mouse antisera (Fig. 7A) and all seven were killed by therabbit antisera (Fig. 8A). The rabbit antisera also mediatedphagocytic killing (50%) of several serogroup-heterologousstrains (Fig. 8B), but the mouse antisera had no killing activity50% against serogroup-heterologous strains (Fig. 7B). Curi-ously, the E. coli HB101 mouse antiserum killed two P. aerugi-nosa O11 strains, 6073 and 6206, while the rabbit antiserum toE. coli HB101 showed minimal killing of these P. aeruginosastrains. This cross-reactive opsonic killing was likely due to ashared epitope, which was not further characterized.

The level of opsonophagocytic killing activity elicited afteri.n. immunization of mice with the live, attenuated strainPAO1�aroA was compared with that generated after i.n. im-munization with heat-killed PAO1 (Fig. 9). For 5 of the 7prototypic P. aeruginosa serogroup O2/O5 strains, opsonic kill-ing activity engendered by i.n. immunization with PAO1�aroAwas significantly higher than that with heat-killed PAO1. Kill-ing of the homologous strain PAO1 was similar for the twovaccines, while killing of Fisher IT-7 was better after the heat-killed vaccine. Similarities between PAO1 and Fisher IT-7 areexpected, since PAO1 is classified as an IT-7 strain when se-

FIG. 6. Opsonophagocytic killing of PAO1 by dilutions of mouseand rabbit antisera raised against PAO1�aroA after i.n. immunization.Bars represent mean percent killing of two to four replicates relative tono-serum control, and error bars represent the standard errors of themeans. Negative killing indicates growth during the 90-min incubation.Rabbit antiserum raised against heat-killed Fisher IT-7 by intravenousimmunization served as a positive control.



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rogrouping is performed with antisera to the prototypic FisherIT strains.

A substantial proportion of the IgG elicited by i.n. immuni-zation with PAO1�aroA was directed against the LPS of P.aeruginosa, as indicated by comparing IgG titers to whole cellsof the parental strain PAO1, AK44 (an O-antigen-deficient,complete-LPS-core mutant of PAO1), and AK1012 (an O-antigen-deficient, incomplete-LPS-core mutant of PAO1) (Fig.10A). The differences in titers against AK44 between themouse and rabbit antisera suggest that the mouse antiserumpossesses relatively low amounts of antibody directed againstthe LPS outer core, while the rabbit antiserum has a highproportion of outer-core-directed antibody. Adsorption of therabbit antisera with the isogenic O-antigen-deficient P. aerugi-nosa strain AK44 or with the O-antigen-deficient serogroupO3 strain PAC557 did not affect the level of phagocytic killingof PAO1, while adsorption with PAO1 reduced killing to15%, thereby confirming the importance of antibody to theO antigen in phagocytic killing (Fig. 10B).

DISCUSSION

For over 30 years, it has been clear that high-level immunityto P. aeruginosa infections can be mediated by antibodies to theLPS O antigen (13). However, just as carbohydrate-based vac-cines for other bacterial pathogens have been problematic, itappears that protective epitopes are poorly immunogenic while

nonprotective or minimally cross-protective O-antigen epi-topes provoke the best immune responses in preclinical eval-uations of vaccines for mice and rabbits (19). Thus, the poorimmunogenicity of the key antigenic determinants of P. aerugi-nosa LPS O antigens may lie at the core of the lack of effectiveLPS-specific vaccines. In addition, with recent evidence frommultiple investigators that P. aeruginosa readily enters lung andcorneal epithelial cells during infection (14, 21, 46), a potentialrole for cell-mediated immunity is clear, indicating that in spiteof the strong role played by antibodies to LPS in protectiveimmunity, they may not be sufficient to control infections.Indeed, T-cell-based immune protection against P. aeruginosainfections has been demonstrated by several groups over thepast 15 years (9, 29, 35–39).

Other P. aeruginosa vaccine strategies have focused onflagellar antigens (24), on outer membrane proteins F (34, 41)and I (34), and more recently, on the PcrV antigen componentof the type III secretion system (54). While protection againstheterologous serogroups has sometimes been seen, the protec-tion afforded by these non-LPS-based vaccines has, as a rule,been of only modest potency. A particularly striking illustra-tion of the remarkable protective efficacy of LPS-based vac-cines is a study in which an outer membrane protein F vaccineprotected 30 to 95% of burned mice against challenge doses upto 2 � 106 CFU of six different serogroups while an LPS-basedvaccine protected against a challenge dose of 3 � 1011 CFU ofthe homologous strain (40).

FIG. 7. Opsonophagocytic killing of P. aeruginosa serogroup O2/O5 (A) and heterologous (B) strains by a 1:8 dilution of antisera fromPAO1�aroA-immunized or E. coli HB101-immunized (control) C3H/HeN mice. The International Antigenic Typing Scheme (IATS) (31, 32)serogroup of each heterologous strain is indicated below the strain designation. Bars represent mean percent killing of two to four replicatesrelative to no-serum control, and error bars represent the standard errors of the means. Negative killing indicates growth during the 90-min incubation.Rabbit antisera raised by intravenous immunization with heat-killed whole bacteria from the same serogroup served as positive controls. Tubes withPAO1�aroA antiserum but without PMNs served as additional negative controls. �, P 0.001; #, P 0.05 by ANOVA with Fisher’s PLSD forcomparison with E. coli HB101 antiserum., P 0.01 by ANOVA with Fisher’s PLSD for comparison with PAO1�aroA antiserum.



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Mutations in the aroA gene, which encodes an enzyme es-sential for the synthesis of aromatic amino acids (5-enolpyru-vylshikimate 3-phosphate synthase of the shikimate pathway),have been utilized with several other pathogens, including Sal-monella species (58) and Aeromonas hydrophila (22), for theproduction of live, attenuated vaccine strains. In fact, in theSalmonella enterica serovar Typhimurium system, aroA dele-tion mutants have been used as delivery vehicles to vaccinatemice against plasmid-encoded foreign proteins, with subse-quent generation of broad cellular immunity (33, 49). Al-though single aroA deletion mutants in Salmonella entericaserovar Typhi retain sufficient virulence to make them unac-ceptable as human vaccines, the intrinsically lower virulence ofP. aeruginosa was predicted to allow single aroA deletion mu-tants to be sufficiently attenuated to permit study in animalmodels.

In the present study, we have constructed an unmarked aroAdeletion mutant of the common laboratory strain of P. aerugi-nosa, PAO1, and confirmed that it is auxotrophic for aromaticamino acids. The strain is highly attenuated in mice in thatdoses up to 5 � 109 CFU can be given i.n. or i.p. without anyapparent adverse effects. We have previously shown that nasalapplication of a bacterial inoculum in anesthetized adult miceresults in rapid translocation of two-thirds or more of the

FIG. 8. Opsonophagocytic killing of P. aeruginosa serogroup O2/O5 (A) and heterologous (B) strains by a 1:8 dilution of serum fromPAO1�aroA-immunized or E. coli HB101-immunized (control) rabbits. The International Antigenic Typing Scheme (IATS) (31, 32) serogroup ofeach heterologous strain is indicated below the strain designation. Bars represent mean percent killing of two to four replicates relative to no-serumcontrol, and error bars represent the standard errors of the means. Negative killing indicates growth during the 90-min incubation. Rabbitantiserum raised by intravenous immunization with heat-killed whole bacteria from the same serogroup served as positive controls. Tubes withPAO1�aroA antiserum but without PMNs served as additional negative controls. �, P 0.001; #, P 0.05 by ANOVA with Fisher’s PLSD forcomparison with E. coli HB101 antiserum.

FIG. 9. Opsonophagocytic killing of P. aeruginosa serogroup O2/O5 strains by sera at the indicated dilutions from C3H/HeN miceimmunized i.n. with either PAO1�aroA or heat-killed PAO1. Barsrepresent mean percent killing of two to four replicates relative tono-serum control, and error bars represent the standard errors of themeans. �, P 0.001 by ANOVA with Fisher’s PLSD in comparisonwith antiserum from mice immunized with PAO1�aroA; #, P 0.001by ANOVA with Fisher’s PLSD in comparison with antiserum frommice immunized with heat-killed PAO1.



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inoculum into the lungs (1). When virulent strains of P. aerugi-nosa are administered, the mice die of pneumonia and sys-temic spread within 72 h. In this murine lung infection model,the aroA deletion mutant was translocated to the lungs lesswell (only about 0.1% of the inoculum could be recovered fromthe lungs immediately after application) and was cleared fromthe lung by 4 days after inoculation. Notably, there was nodissemination to the blood, liver, or spleen; and there was onlylow-level inflammation in lungs as determined by histologicalevaluation at 48 h. The amount of internalized bacteria as apercentage of total bacteria recovered from the lungs wasessentially 100% by 18 h after inoculation with the aroA dele-tion mutant strain. By comparison, the percentage of internal-ized bacteria in this model after challenge with the parentalstrain PAO1 is approximately 10% at most time points (1).This propensity of the aroA deletion mutant for intracellularlocalization is likely due to its inability to survive in an extra-cellular environment lacking aromatic amino acids. The lim-ited but significant survival of the attenuated strain is alsoimportant for the prospect of using engineered versions of thearoA deletion mutant to overexpress protein antigens thatmight bolster the immune response.

We have found that i.n. immunization of mice and rabbitswith the aroA deletion mutant of PAO1 elicits high titers ofIgG against whole cells and boiled cells of multiple subtype-and even serogroup-heterologous strains of P. aeruginosa.These high titers were achieved without the use of adjuvants.While IgG titers determined by ELISA are useful in screeningsera for possible protective serologic responses, the levels ofopsonic antibodies against P. aeruginosa are the best predictorsof protective efficacy in animal models. Along these lines, it isremarkable that i.n. immunization with a single aroA deletionmutant strain engenders opsonic antibodies against the paren-tal strain as well as multiple strains within serogroup O2/O5.This is in stark contrast to our previous findings that i.p. im-munization of mice with the purified high-molecular-weightO-polysaccharide from PAO1 elicited low-level opsonic titersagainst only two of the prototypic serogroup O2/O5 strainsand did not elicit a good opsonic antibody response even tothe parental strain (19). Furthermore, in challenge experi-ments using the mouse model of pneumonia and systemicspread after i.n. inoculation (1), mice immunized i.n. withPAO1�aroA were 100% protected from death while 100% ofmice immunized i.n. with E. coli HB101 died within 4 days ofchallenge with a cytotoxic variant of PAO1 (PAO1 transfectedwith a plasmid expressing the cytotoxin ExoU and its chaper-one [1]) at a dose 100-fold higher than the 50% lethal dose(J. Goldberg, M. Brinig, M. Grout, K. Hatano, F. Coleman, G.Priebe, and G. Pier, Abstr. 100th Gen. Meet. Am. Soc. Micro-biol., abstr. D-155, 2000).

We also compared the i.n. immunization of mice with heat-killed PAO1 to that with PAO1�aroA and found that the liveattenuated strain PAO1�aroA generated significantly higherlevels of opsonic antibody against five of the seven prototypicP. aeruginosa serogroup O2/O5 strains (Fig. 9). This may bedue to the ability of the live attenuated strain to serve as abetter immunogen than heat-killed bacteria by means of sim-ple multiplication as well as by exploitation of the naturalpathways of infection, especially the intracellular phase. Pre-sumably, modifications of bacterial antigens that occur during

FIG. 10. Antibodies elicited by i.n. immunization with PAO1�aroAare directed against the LPS of P. aeruginosa. (A) IgG titers of mouseand rabbit antisera to PAO1�aroA against P. aeruginosa PAO1, AK44(an O-antigen-deficient, complete-LPS-core mutant of PAO1), andAK1012 (an O-antigen-deficient, incomplete-LPS-core mutant ofPAO1). Bars represent endpoint titers as determined by linear regres-sion, and error bars indicate the 95% confidence intervals. (B) Ad-sorption of rabbit antiserum to PAO1�aroA with the O-antigen-defi-cient mutants AK44 and PAC557 does not alter the opsonic activity,while adsorption with PAO1 abolishes opsonic activity. Bars representmean percent killing of two to four replicates relative to no-serumcontrol, and error bars represent the standard errors of the means.Negative killing indicates growth during the 90-min incubation. Tubeswith the variously adsorbed PAO1�aroA antisera but without PMNsserved as negative controls. �, P 0.0001 by ANOVA with Fisher’sPLSD for comparisons with either unadsorbed, AK44-adsorbed, orPAC557-adsorbed antiserum.



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in vivo infection would also occur in the live attenuated strainand could thereby engender a broader immune response. Ourfindings that the opsonic activity was not removed by adsorp-tion with the O-antigen-deficient strains AK44 and PAC557(Fig. 10) indicate, as expected, that the active antibodies afteri.n. immunization with PAO1�aroA are directed against O-antigen epitopes of LPS.

Thus, i.n. immunization of mice and rabbits with the singlestrain PAO1�aroA elicits opsonic antibodies against multiplemembers of the P. aeruginosa serogroup O2/O5, indicating thesignificant potential of using live, attenuated strains for vacci-nation against LPS-smooth strains of P. aeruginosa.

ACKNOWLEDGMENTS

This work was supported by NIH grants AI22535 (G.B.P.), AI50036(G.P.P.), and AI37632 (J.B.G.), by the University of Virginia School ofMedicine and a Student Traineeship Grant from the Cystic FibrosisFoundation (BRINIG99H0) (M.M.B.), and by the Anesthesia Depart-ment of Children’s Hospital and Harvard Medical School, Boston,Mass. (G.P.P.).

We thank Vincent Carey for assistance with statistical analysis.

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