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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.

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.

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.

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.

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.

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.

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.

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.

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.