DNA vaccines against chronic lung infections by Pseudomonas aeruginosa.

Vaccines containing outer membrane protein F (OprF) of Pseudomonas aeruginosa are effective in reducing lesion severity in a mouse pulmonary chronic infection model. One OprF-based vaccine, called F/I, contains carboxy oprF sequences fused to oprI in an expression vector. When delivered three times biolistically by gene gun, the F/I vaccine induces protection that is antibody-mediated in outbred mice. To demonstrate the role of F/I-induced antibody-mediated immunity, B-cell-deficient [B(-)] and B-cell-intact [B(+)] mice were immunized with F/I, challenged with Pseudomonas, and examined for lesion severity. As expected, F/I-immunized B(+) mice had fewer and less severe lesions than vector-immunized B(+) mice. However, surprisingly, F/I- and vector-immunized B(-) mice were equally protected to levels similar to F/I-immunized B(+) mice. Examination of immune cell populations and cytokine levels indicated a relative increase in the quantity of CD3+ T-lymphocytes in vector- or F/I-immunized and challenged B(-) mice compared to B(+) mice. These data indicate the protective role played by cell-mediated immunity in B(-) mice, which supports our hypothesis that cell-mediated immunity can play an important role in protection against P. aeruginosa.

Protection against Pseudomonas aeruginosa chronic lung infection in mice by genetic immunization against outer membrane protein F (OprF) of P. aeruginosa.

The Pseudomonas aeruginosa major constitutive outer membrane porin protein OprF, which has previously been shown to be a protective antigen, was targeted as a DNA vaccine candidate. The oprF gene was cloned into plasmid vector pVR1020, and the plasmid vaccines were delivered to mice by biolistic (gene gun) intradermal inoculation. Antibody titers in antisera from immunized mice were determined by enzyme-linked immunosorbent assay, and the elicited antibodies were shown to be specifically reactive to OprF by immunoblotting. The immunoglobulin G (IgG) immune response was predominantly of the IgG1 isotype. Sera from DNA vaccine-immunized mice had significantly greater opsonic activity in opsonophagocytic assays than did sera from control mice. Following the initial immunization and two consecutive boosts, each at 2-week intervals, protection was demonstrated in a mouse model of chronic pulmonary infection by P. aeruginosa. Eight days postchallenge, both lungs were removed and examined. A significant reduction in the presence of severe macroscopic lesions, as well as in the number of bacteria present in the lungs, was seen. Based on these findings, genetic immunization with oprF has potential for development as a vaccine to protect humans against infection by P. aeruginosa.

Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection.

Outer membrane protein F of Pseudomonas aeruginosa has vaccine efficacy against infection by P. aeruginosa as demonstrated in a variety of animal models. Through the use of synthetic peptides, three surface-exposed epitopes have been identified. These are called peptides 9 (aa 261-274 in the mature F protein, TDAYNQKLSERRAN), 10 (aa 305-318, NATAEGRAINRRVE), and 18 (aa 282-295, NEYGVEGGRVNAVG). Both the peptide 9 and 10 epitopes are protective when administered as a vaccine. In order to develop a vaccine that is suitable for use in humans, including infants with cystic fibrosis, the use of viral vector systems to present the protective epitopes has been investigated. An 11-amino acid portion of epitope 10 (AEGRAINRRVE) was successfully inserted into the antigenic B site of the hemagglutinin on the surface of influenza virus. This chimeric influenza virus protects against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Attempts to derive a chimeric influenza virus carrying epitope 9 have been unsuccessful. A chimeric plant virus, cowpea mosaic virus (CPMV), with epitopes 18 and 10 expressed in tandem on the large coat protein subunit (CPMV-PAE5) was found to elicit antibodies that reacted exclusively with the 10 epitope and not with epitope 18. Use of this chimeric virus as a vaccine afforded protection against challenge with P. aeruginosa in the mouse model of chronic pulmonary infection. Chimeric CPMVs with a single peptide containing epitopes 9 and 18 expressed on either of the coat proteins are in the process of being evaluated. Epitope 9 was successfully expressed on the coat protein of tobacco mosaic virus (TMV), and this chimeric virus is protective when used as a vaccine in the mouse model of chronic pulmonary infection. However, initial attempts to express epitope 10 on the coat protein of TMV have been unsuccessful. Efforts are continuing to construct chimeric viruses that express both the 9 and 10 epitopes in the same virus vector system. Ideally, the use of a vaccine containing two epitopes of protein F is desirable in order to greatly reduce the likelihood of selecting a variant of P. aeruginosa that escapes protective antibodies in immunized humans via a mutation in a single epitope within protein F. When the chimeric influenza virus containing epitope 10 and the chimeric TMV containing epitope 9 were given together as a combined vaccine, the immunized mice produced antibodies directed toward both epitopes 9 and 10. The combined vaccine afforded protection against challenge with P. aeruginosa in the chronic pulmonary infection model at approximately the same level of efficacy as provided by the individual chimeric virus vaccines. These results prove in principle that a combined chimeric viral vaccine presenting both epitopes 9 and 10 of protein F has vaccine potential warranting continued development into a vaccine for use in humans.

Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa.

A chimeric tobacco mosaic virus (TMV) was constructed by inserting sequences representing peptide 9-14mer (TDAYNQKLSERRAN) of outer membrane (OM) protein F of Pseudomonas aeruginosa between amino acids Ser154 and Gly155 of the TMV coat protein (CP). This is the first example of TMV being used to construct a chimera containing a bacterial epitope. Mice immunized with TMV-9-14 produced anti-peptide-9-14mer-specific antibodies that reacted in whole-cell ELISA with all seven Fisher-Devlin (FD) immunotype strains of P. aeruginosa, reacted specifically by Western blotting with OM protein F extracted from all seven FD immunotypes, and were opsonic in opsonophagocytic assays. The chimeric TMV-9-14 vaccine afforded immunoprotection against challenge with wild-type P. aeruginosa in a mouse model of chronic pulmonary infection. TMV-9-14 is an excellent candidate for further development as a vaccine for possible use in humans to protect against P. aeruginosa infections.

Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus.

A synthetic peptide (peptide 10) representing a surface-exposed, linear B cell epitope from outer-membrane (OM) protein F of Pseudomonas aeruginosa was shown previously to afford protection in mice from P. aeruginosa infection. This peptide was expressed in tandem with the protein F peptide 18 on each of the two coat proteins of cowpea mosaic virus (CPMV). The chimaeric virus particles (CVPs) expressing the peptides on the S (small) coat protein (CPMV-PAE4) and L (large) coat protein (CPMV-PAE5) were used to immunize mice. Following subcutaneous immunization in Freund's and QuilA adjuvants, CPMV-PAE4 induced antibodies predominantly against peptide 18, whereas CPMV-PAE5 produced antibodies exclusively against peptide 10, indicating that the site of peptide expression on CPMV influences its immune recognition. The anti-peptide antibodies elicited by CPMV-PAE5 were predominantly of the IgG2a isotype, indicating a highly polarized TH1-type response. The peptide-specific IgG2a strongly recognized the whole F protein, but more importantly, recognized protein F in all seven Fisher-Devlin immunotypes of P. aeruginosa. Furthermore, the peptide-specific IgG2a in CVP/QS-21 adjuvant-immunized mice was shown to bind complement and to augment phagocytosis of P. aeruginosa by human neutrophils in vitro. The ability of CPMV-PAE5 to induce P. aeruginosa-specific opsonic IgG2a gives it potential for further development as a protective vaccine against P. aeruginosa.

A chimeric influenza virus expressing an epitope of outer membrane protein F of Pseudomonas aeruginosa affords protection against challenge with P. aeruginosa in a murine model of chronic pulmonary infection.

The ability of a chimeric influenza virus containing, within the antigenic B site of its hemagglutinin, an 11-amino-acid (AEGRAINRRVE) insert from the peptide 10 epitope of outer membrane (OM) protein F of Pseudomonas aeruginosa to serve as a protective vaccine against P. aeruginosa was tested by using the murine chronic pulmonary infection model. Mice immunized with the chimeric virus developed antibodies that reacted in an enzyme-linked immunosorbent assay with peptide 10, with purified protein F, and with whole cells of various immunotype strains of P. aeruginosa but failed to react with a protein F-deficient strain of P. aeruginosa. The chimeric-virus antisera reacted specifically with protein F alone when immunoblotted against proteins extracted from cell envelopes of each of the seven Fisher-Devlin immunotype strains and had significantly greater in vitro opsonic activity for P. aeruginosa than did antisera from wild-type influenza virus-immunized mice. Subsequent to intratracheal challenge with agar-encased cells of P. aeruginosa, chimeric-virus-immunized mice developed significantly fewer severe lung lesions than did control mice immunized with the wild-type influenza virus. Furthermore, the chimeric influenza virus-immunized group had a significantly smaller percentage of mice with >5 x 10(3) CFU of P. aeruginosa in their lungs upon bacterial quantitation than did the control group. These data indicate that chimeric influenza viruses expressing epitopes of OM protein F warrant continued development as vaccines to prevent pulmonary infections caused by P. aeruginosa.

Low-frequency electromagnetic fields alter the replication cycle of MS2 bacteriophage.

The effect of exposure to 60-Hz electromagnetic fields (EMFs) on RNA coliphage MS2 replication was studied. EMF exposure commenced when the bacterial cultures were inoculated with the phage (t = 0). In 12 experiments in which the strength of the field was 5 G, a significant delay in phage yield was found in the EMF-exposed cultures 45-65 min after inoculation, compared with control cultures. However, the EMF did not alter the final phage concentration. Experiments at 25 G (N = 5) suggested that the stronger field resulted in both impeded phage replication and increased phage yield. No differences between test groups were found in experiments involving sham-EMF exposure, thereby indicating that the results obtained with the EMFs were not due to systematic error. It appears that MS2, which codes for only four proteins, is the simplest biological system in which an EMF-induced effect has been demonstrated. The MS2 system is, therefore, conducive to follow-up studies aimed at understanding the level and nature of the underlying interaction process, and perhaps to biophysical modeling of the interaction process.

Chimeric influenza viruses incorporating epitopes of outer membrane protein F as a vaccine against pulmonary infection with Pseudomonas aeruginosa.

Peptide 10 (NATAEGRAINRRVE, residues 305-318 of mature protein F) is one of two linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa both of which have been shown to elicit whole cell-reactive antibodies and to afford protection in animal models against P. aeruginosa infection. Influenza A virus was chosen as a vector to present this epitope in a human-compatible vaccine. Various lengths of the peptide 10 epitope ranging from a 5-mer (GRAIN), 7-mer (AINRRVE), 8-mer (TAEGRAIN), 9-mer (GRAINRRVE), 11-mer (AEGRAINRRVE) to a 12-mer (TAEGRAINRRVE) were attempted to be presented into the antigenic B-site of the hemagglutinin (HA) of live recombinant influenza virus. Using PCR, DNA sequences encoding these various peptide 10 lengths were inserted into the HA gene of influenza A/WSN/33 virus. By using a reverse-genetics transfection system, RNA transcribed in vitro from these chimeric HA genes was reassorted into infectious virus. To date chimeric viruses have been rescued and purified containing the peptide 10 5-mer, 7-mer, 8-mer, and 11-mer. RT-PCR and sequencing have confirmed the presence of P. aeruginosa sequences in the HA RNA segment of each chimeric virus. Each of the four chimeric viruses produced to date was used to immunize mice to determine the ability of each chimeric virus to elicit antibodies reactive with whole cells of P. aeruginosa. The immunization protocol consisted of a series of three intranasal inoculations, followed by two intramuscular injections of the chimeric virus. The chimeric virus incorporating the 11-mer elicited IgG antibodies that reacted with various immunotype strains of P. aeruginosa in a whole cell ELISA at titers of 80 to 2,560, whereas the chimeric virus incorporating the 8-mer elicited whole cell-reactive IgG antibodies at titers of 320 to 2,560. These data suggest that these two chimeric viruses may have vaccine efficacy against P. aeruginosa infection. These studies may result in the development of a chimeric influenza virus-protein F vaccine which would prove to be suitable for use in children with cystic fibrosis for the prevention of pulmonary colonization of these children with P. aeruginosa.

Use of synthetic peptides to identify surface-exposed, linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa.

In a previous study (Hughes EE, Gilleland LB, Gilleland HE Jr. [1992] Infect Immun 60:3497-3503), ten synthetic peptides were used to test for surface-exposed antigenic regions located throughout the length of outer membrane protein F of Pseudomonas aeruginosa. An additional nine peptides of 11-21 amino acid residues in length were synthesized. Antisera collected from mice immunized with each of the 19 synthetic peptides conjugated to keyhole limpet hemocyanin were used to determine which of the peptides had elicited antibodies capable of reacting with the surface of whole cells of the various heterologous Fisher-Devlin immunotypes of P. aeruginosa. Cell surface reactivity was measured by an enzyme-linked immunosorbent assay (ELISA) with whole cells of the various immunotypes as the ELISA antigens and by opsonophagocytic uptake assays with the various peptide-directed antisera, immunotype 2 P. aeruginosa cells, and polymorphonuclear leukocytes of human and murine origin. Three peptides located in the carboxy-terminal portion of protein F elicited antibodies with the greatest cell-surface reactivity. Peptide 9 (TDAYNQKLSERRAN), peptide 10 (NATAEGRAINRRVE), and peptide 18 (NEYGVEGGRVNAVG) appear to have sufficient potential for further development as vaccine candidates for immunoprophylaxis against infections caused by P. aeruginosa. A topological model for the arrangement of protein F within the outer membrane of P. aeruginosa is presented.

Synthetic peptides representing two protective, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa elicit whole-cell-reactive antibodies that are functionally pseudomonad specific.

Peptide 9 (TDAYNQKLSERRAN) and peptide 10 (NATAEGRAINRRVE) represent surface-exposed epitopes of outer membrane protein F of Pseudomonas aeruginosa. Rats immunized with four intramuscular inoculations on days 0, 14, 28, and 42 with either peptide 9 or peptide 10 conjugated to keyhole limpet hemocyanin were afforded protection against pulmonary lesions when examined 7 days subsequent to challenge (day 56) via intratracheal inoculation of P. aeruginosa-containing agar beads. Peptide 9 shares considerable homology with other OmpA-related outer membrane proteins in various bacteria, whereas peptide 10 displays little homology with these other proteins. Antisera directed to peptide 9 reacted weakly with cell envelope proteins from the various other OmpA-associated bacteria upon immunoblot analysis. However, antisera directed to peptide 10 reacted only with Neisseria gonorrhoeae cell envelope proteins upon immunoblot analysis. Antisera to both peptides 9 and 10 reacted at minimal titers with whole cells of the various other bacteria in a whole-cell enzyme-linked immunosorbent assay (ELISA) but antisera to each of the peptides reacted at high titers when various strains of P. aeruginosa were used as the ELISA antigen. Antibodies to peptides 9 and 10 were protective, reactive to all strain of P. aeruginosa tested except for a protein F-deficient mutant, and functionally specific against pseudomonads.

Preservation of pulmonary function by an outer membrane protein F vaccine. A study in rats with chronic pulmonary infection caused by Pseudomonas aeruginosa.

This study investigated the ability of a protein F vaccine to reduce macroscopic evidence of lung damage and preserve pulmonary function in immunized animals in a rat model of chronic pulmonary infection caused by Pseudomonas aeruginosa. Other membrane protein F of P aeruginosa was purified by extraction from polyacrylamide gels of cell envelope proteins of the PAO1 immunotype 7 strain. Rats were immunized intramuscularly with either 25 micrograms of the purified protein F or bovine serum albumin on days 0 and 14 and then challenged on day 28 via intratracheal inoculation of agar beads containing cells of an immunotype 3 clinical isolate of P aeruginosa. Also, included was a noninfected control group which received only sterile agar beads. On day 35, the lungs were excised, pulmonary compliance measured, and the lungs examined macroscopically for the presence and severity of lesions. The protein F-immunized rats had a significant (p < 0.01) reduction in the number of severe pulmonary lesions as compared with bovine serum albumin-immunized rats. Lung compliance (CL) was significantly (p < 0.001) reduced in rats which were immunized with bovine serum albumin (n = 17, CL = 0.12 +/- 0.008), whereas CL of protein F-immunized rats (n = 12, CL = 0.17 +/- 0.006) was similar to that of noninfected control rats (n = 5, CL = 0.15 +/- 0.008). This study demonstrated that a protein F vaccine has the ability to decrease macroscopic lung lesions from infection and preserve pulmonary function in actively immunized rats upon subsequent challenge with P aeruginosa in this model of chronic lung infection.

Vaccine efficacies of elastase, exotoxin A, and outer-membrane protein F in preventing chronic pulmonary infection by Pseudomonas aeruginosa in a rat model.

The protective efficacies of eight vaccine preparations consisting of Pseudomonas aeruginosa outer-membrane protein F, elastase and exotoxin A toxoid, administered either individually or in various combinations, were determined in a rat model of chronic pulmonary infection. Rats were immunised intramuscularly at 2-week intervals (days 0, 14 and 28). On day 42, blood was collected and antisera were obtained from each vaccine group for use in an enzyme-linked immunosorbent assay which determined the titre of IgG antibodies elicited by each vaccine. Also on day 42, rats were challenged by intratracheal inoculation of a clinical isolate of P. aeruginosa encased within agar beads. On day 49, the animals were killed and the lungs were examined macroscopically for the presence of lesions and fixed for histological examination. When compared with control rats immunised with bovine serum albumin, rats immunised with protein F alone as a vaccine received significant protection against the development of severe pulmonary lesions. Elastase used alone as a vaccine provided some protection against severe lung lesions and reduced the incidence of microscopic peribronchial inflammation. However, the combination of protein F plus elastase as a vaccine did not afford protection from severe lesions, and there was an increased incidence of necrotising granulomas in the lungs from this vaccine group. Protection against lung lesions from the three-component vaccine consisting of protein F, elastase and exotoxin A toxoid was similar to that provided by the protein F vaccine. Neither macroscopic nor histological evidence showed any enhancement of protective efficacy for the three-component vaccine over that of the protein F vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Neutrophil-mediated nitrosamine formation: role of nitric oxide in rats.

It is well known that chronic inflammation of the colon and rectum is associated with an increased risk of colorectal cancer, but the mechanisms by which inflammation promotes neoplasia remain undefined. The authors propose that inflammatory neutrophils may produce carcinogenic nitrosamines via the L-arginine-dependent formation of nitrogen oxides such as nitric oxide. Therefore, the objectives of the study were to characterize the L-arginine-dependent formation of nitrogen oxides by inflammatory (elicited) neutrophils using conditions that more closely mimic the extravascular (i.e., interstitial) compartment of the gut and to characterize the neutrophil-dependent N-nitrosation of a model amine to yield its nitrosamine derivative. In the absence of any metabolic activation, adherent, inflammatory neutrophils (2 x 10(6) cells) produced 12.8 +/- 1.4 mumol/L of nitrite during a 4-hour incubation period. Omission of L-arginine and/or inhibition of nitric oxide synthase by the addition of 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) resulted in 35%-78% inhibition of nitrite production, suggesting that nitrite was derived from nitric oxide. By comparison, neither circulating rat neutrophils nor elicited rat macrophages produced significant amounts of nitrite under the same conditions. Furthermore, elicited neutrophils (2 x 10(6) cells) were capable of N-nitrosating 2,3-diaminonaphthalene to yield its nitrosamine derivative 1-naphtho-2,3-triazole (282 +/- 12 nmol/L) in a time- and cell-dependent pattern similar to that of nitrite production. Addition of a variety of antioxidants (e.g., ascorbic acid, reduced glutathione, alpha-tocopherol analog), 5-aminosalicylic acid, or L-NAME resulted in 80%-85% inhibition of neutrophil-mediated nitrosamine formation. Taken together, these data suggest that inflammatory neutrophils may represent an important metabolic source of endogenous carcinogens during times of active intestinal inflammation.

Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa.

By using the published amino acid sequence for mature outer membrane protein F of Pseudomonas aeruginosa, a computer-assisted analysis was performed to identify sites with potential as surface-exposed, antigenic regions located throughout the length of the protein molecule. Synthetic peptides 13 to 15 amino acid residues in length were synthesized for 10 such regions. Mice were immunized with each of the 10 synthetic peptides conjugated to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) of the antisera was performed by using each of the synthetic peptides as the ELISA antigen to verify that immunoglobulin G (IgG) antibodies capable of reacting with the peptide used as immunogen were elicited by each peptide. Each of the antipeptide antisera was screened for the presence of IgG antibodies that could bind to the surface of intact cells of strains representing the seven heterologous Fisher-Devlin immunotypes of P. aeruginosa by use of an ELISA with whole cells of the various strains as the ELISA antigen. Three peptides elicited antibodies capable of reacting with whole cells of all seven immunotype strains. Peptide 10, corresponding to amino acid residues 305 to 318, elicited whole-cell-reactive antibodies at high titers. Peptide 9, corresponding to amino acid residues 261 to 274, elicited whole-cell-reactive antibodies at more intermediate titers. Peptide 7, corresponding to amino acid residues 219 to 232, elicited such antibodies only at low titers. The carboxy-terminal portion of the mature protein appears to be the immunodominant portion. In particular, peptides 10 (NATAEGRAINRRVE) and 9 (TDAYNQKLSERRAN) appear to have potential for use as immunogens in a synthetic vaccine for immunoprophylaxis against infections caused by P. aeruginosa. Antisera from mice immunized with either peptide 9 or 10 mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa.

Adaptive resistance to aminoglycoside antibiotics in Pseudomonas aeruginosa.

Aminoglycoside-resistant variants of Pseudomonas aeruginosa strain PAO1 were readily selected by culturing the organism in medium containing increasing concentrations of gentamicin, tobramycin or amikacin until the strains were growing in a concentration of drug 128-fold greater than the minimal inhibitory concentration for the sensitive parent strain. These resistant strains exhibited characteristics previously associated with the impermeability type of resistance mechanism, i.e., they grew more slowly than the parent strain, the resistance was unstable in the absence of the antibiotic, and adaptation to one of the antibiotics conferred cross-resistance to other aminoglycosides. The adapted strains grew, with minimal morphological alterations, in concentrations of the various aminoglycosides that normally produced cell envelope damage, misshapen and filamentous cell formation, and cell lysis in the sensitive strain. Neither protein H1 nor phospholipid alterations appear to play a significant role in adaptive resistance to aminoglycoside antibiotics in this model system. The acquisition of adaptive resistance to the aminoglycoside antibiotics did not confer resistance to polymyxin B, another cationic antibiotic which is thought to share binding sites within the outer membrane with the aminoglycosides.

Outer membrane protein F preparation of Pseudomonas aeruginosa as a vaccine against chronic pulmonary infection with heterologous immunotype strains in a rat model.

Outer membrane protein F (porin) was purified by extraction from polyacrylamide gels of cell envelope proteins of the Pseudomonas aeruginosa PAO1 strain. Rats were immunized intramuscularly with 25 micrograms of protein F on days 1 and 14 and then challenged on day 28 via intratracheal inoculation of bacterium-containing agar beads. On day 35 the lungs were either fixed for histological examination or submitted for quantitation of the bacteria present. protein F immunization afforded significant protection against challenge with six of six heterologous lipopolysaccharide immunotype strains of P. aeruginosa. By an enzyme-linked immunosorbent assay, the protein F-immunized rats had both immunoglobulin G and M antibody responses to cell envelopes of all six of the heterologous immunotype strains. Protein F immunization greatly enhanced the ability of the rats to clear the inoculated P. aeruginosa from the lungs and significantly reduced the incidence and severity of pulmonary lesions as compared with those in bovine serum albumin-immunized control rats. These data show the efficacy of outer membrane protein F as a protective vaccine in a rat model of chronic pulmonary infection.