AU2007231760B2 - Method and compositions for immunization with the pseudomonas V antigen - Google Patents

Description

METHOD AND COMPOSITIONS FOR IMMUNIZATION WITH THE PSEUDOMONAS V ANTIGEN In the specification and claims the term 'comprising' shall be 5 understood to have a broad meaning similar to the term 'including' and will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. This definition also applies to variations on the term 'comprising' such as 'comprise' and 'comprises'. 10 BACKGROUND OF THE INVENTION Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is capable of causing fatal acute lung infections in critically ill 15 individuals (1). The ability of the bacterium to damage the lung epithelium has been linked with the expression of toxins that are directly injected into eukaryotic cells via a type ll-mediated secretion and translocation mechanism (2,3). The proteins encoded by the P. aeruginosa type Ill secretion and 20 translocation apparatus demonstrate a high level of amino acid identity with members of the Yersinia Yop regulon (4-6). Of all the type Ill systems discovered in Gram-negative bacteria, only P. aeruginosa possesses a homologue to the Yersinia V antigen, PcrV (see 6 for review of type Ill systems). Homologous proteins to the secretion and translocation 25 apparatus are encoded by both plant and animal pathogenic bacteria. These organisms include human pathogens such as Salmonella typhimurium, Shigella flexneri, Enteropathogenic E coli, Chlamydia spp., and plant pathogens such as Xanthamonas campestris, Pseudomonas syringe, Erwinia amylovora and Ralstonia solanacearum. However, only P. 30 aeruginosa and Yersinia encode the V antigen. Yahr, et al., 1997, discloses the sequence of the operon encoding PcrV and compares the sequence to the LcrV protein. Thus, the amino acid -1 sequence of PcrV is known and is available under accession number AF010149 of GenBank. SUMMARY OF THE INVENTION 5 The present invention involves methods and compositions developed from our observation that the Pseudomonas V antigen can be used to protect animals from a lethal lung infection. In one embodiment, the present invention is a method of inhibiting 10 Pseudomonas infection comprising inoculating a patient with an effective amount of PcrV antigen. In another embodiment, DNA encoding PcrV is used as a gene vaccine. In one preferred embodiment, the antigen is expressed as a recombinant protein and used to immunize patients at risk. 15 Preferably, the patient is completely protected from infection. In another embodiment, the DNA encoding PcrV (called pcrV) or a DNA fragment may be used diagnostically to detect P. aeruginosa infection. In another embodiment, the recombinant protein (rPcrV) is used 20 diagnostically to detect antibodies from patients. Patient antibody response to PcrV may be associated with prognosis. Therefore, in this embodiment the recombinant protein is used as a prognostic indicator by measuring the patient's antibody titer. The present invention also provides a method for inhibiting a 25 Pseudomonas infection in an individual by contacting the individual with an effective amount of a PcrV inhibitor, in particular with a PcrV antibody, antibody derivative or fragment, or antibody mimic. PcrV antibodies, antibody derivatives and antibody fragments are also provided. -2- DESCRIPTION OF THE DRAWINGS Figs. 1A and 1 B are a stained gel (Fig. 1A) and Western blot (Fig. 1B) illustrating the phenotypic analysis of PA103ApcrV. 5 Figs. 2A and 2B are a graph (Fig. 2A) and set of bar graphs (Fig. 2B) illustrating the survival and lung injury of P. aeruginosa parental and mutant strains. 10 Figs. 3A and 3B are a graph (Fig. 3A) and a set of bar graphs (Fig. 3B) illustrating the effect of immunization on survival, lung injury, and bacterial colonization. Fig. 4 is a graph of the number of animals surviving a challenge with 15 5 x 105 CFU/mouse of strain PA1 03 after passive administration of polyclonal IgG specific for PcrV, ExoU, PopD or control IgG from an unimmunized animal. Fig. 5 is a graph (Fig. 5A) and a set of bar graphs (Fig. 5B) 20 illustrating survival and protection from lung injury by concomitant administration of IgG to different bacterial antigens and bacterial challenge. One-way ANOVA for lung injury, p=0.026, and lung edema, p < 0.0005. Figs. 6A and B are printouts of SEQ ID NOs:1 and 2 with additional 25 explanatory information. Fig. 6A is SEQ ID NO:1. Fig. 6B is SEQ ID NO:2. -3- Fig. 7 is a printout of SEQ ID NOs:3 and 4 with additional explanatory information. Fig. 8 is a synthetic recombinant single chain antibody (SCFV-M166) (SEQ ID NOs:5 and 6). 5 DESCRIPTION OF THE INVENTION We disclose herein that PcrV has a novel regulatory effect on expression of the type IlIl secreted products, is involved in the translocation of type IlIl toxins, and is the first antigen that protects against lung injury induced by P. aeruginosa infection. Vaccination against PcrV prior to the airspace 10 instillation of anti-PcrV IgG not only ensured the survival of challenged animals but also decreased lung inflammation and injury caused by the bacteria. LcrV, or the V antigen, is a multifunctional protein that regulates secretion/translocation of the Yop effector proteins and plays an extracellular 15 role in pathogenesis by altering the host cytokine response to Yersinia infection (7-11). The only known homologue of this critical pathogenic factor is an extracellular protein encoded by P. aeruginosa, termed PcrV. One embodiment of the present invention is a method of moderating or inhibiting a Pseudomonas infection by immunizing a patient with an effective 20 amount of the PcrV antigen. By "effective amount" we mean an amount of PcrV antigen effective to show some moderation or inhibition of Pseudomonas infection as compared to control subjects or animals who have not been treated with the antigen. By "moderating" we mean that infection is inhibited by at least fifty 25 percent compared to a non-immunized animal. Preferably, infection is completely prevented. A quantitative assessment of infection would preferably include the examination of the amount of bacteria in the -4bloodstream or pleural fluids and/or an examination of lung injury parameters. For example, the absence of bacteria in the bloodstream or pleural fluids would indicate prevention of infection. A reduction in lung injury parameters would indicate that infection is moderated. 5 Infection could be quantitatively assessed by several other clinical indicators, including the reduction of bacterial load in the sputum, blood or pleural fluids, reduction in the size of the infiltrate, oxygenation improvement, reduction in the length of time on mechanical ventilation, reduction in fever and reduction in white blood cell count. 10 By "PcrV antigen" we mean that portion or fragment of the PcrV protein that is necessary to invoke an immune response which prevents or moderates infection. We have used the full-length PcrV protein as an antigen to induce protection. Additionally, we have mapped the protective epitope to the fragment comprising amino acids 144-257 of PcrV. To define the epitope, 15 monoclonal antibodies that protected against infection and cytotoxicity were tested for binding to progressively smaller forms of recombinant PcrV. (By "recombinant PcrV" or "rPcrV" we mean the protein produced from a PcrV gene that has been placed in a non-native host.) This protection localized the region. 20 The PcrV antigen may be most easily obtained by the method we used, commercially available bacterial expression plasmid petl6b from Novagen. The pcrV gene was first cloned from the P. aeruginosa chromosome as part of an operon. The coding region was amplified and inserted into two different vectors. One vector is for expression from P. 25 aeruginosa as shown in Fig. 1. This is a vector from Herbert Schweizer (reference 19) which we modified to contain an appropriate promoter sequence such that PcrV expression is coordinately regulated with the rest of -5the delivery and intoxication apparatus of the bacterium. The second plasmid, pET16b, is for expression and purification purposes from E. coli. The advantage of this system is that we do not have to worry about contaminating P. aeruginosa proteins, the protein is produced in great 5 abundance, and there is a one-step purification process. In this situation the PcrV coding region is amplified to be cloned in frame with a histidine tag provided on the pET16b vector. The multiple histidine residues fused to the amino terminus of PcrV allow affinity chromatography using a nickel-NTA column. Therefore, a preferable PcrV antigen is a recombinant version of the 10 natural PcrV protein. Immunization may be done systemically or intranasally. Immunization of these individuals would preferably start during normal vaccination procedures for other childhood diseases. We would predict long-lived protection with booster doses probably around ages 5 and 10. 15 In another embodiment, one would use DNA encoding the PcrV protein or the complement of this DNA to diagnostically detect P. aeruginosa infection. One would obtain the DNA sequence of the PcrV antigen at GenBank AF010149. The coding region for PcrV is at nucleotides 626-1510. One may also choose to use a fragment of this coding region or complement 20 of this fragment. A successful probe is one that will hybridize specifically to the PcrV DNA and not to other regions. One would preferably use a hybridization probe of at least 40 continuous nucleotides within the antigen sequence or two primers of at least 25 continuous nucleotides within the sequence. One skilled in the art would 25 appreciate that many standard forms of nucleic acid diagnostic techniques would be suitable, for example, hybridization of the single-stranded 40 nucleotide probe to DNA or RNA extracted from a patient's sputum. In another example, patient's sputum would be used as a source for bacterial -6- DNA or RNA to serve as a template for the PCR or RT-PCR reaction, respectively. One would also determine Pseudomonas aeruginosa infection in an individual by contacting a sample obtained from the individual with an 5 antibody specific for PcrV and correlating enhanced antibody binding as compared with a control sample with Pseudomonas aeruginosa infection in the individual. In an additional embodiment, the DNA encoding PcrV is used as a gene vaccine using standard molecular biological methods. For example, 10 one could review the following references for techniques known to those of skill in the art: Davis, H.L., et al., "DNA vaccine for hepatitis B: Evidence for immunogenicity in chimpanzees and comparison with other vaccines," Proc. Nati. Acad. Sci. 93:7213-7218, 1996; Barry, M.A., et a., "Protection against mycoplasma infection using expression-library immunization," Nature 15 377:632-635, 1995; Xiang, Z.Q., et a., "Immune responses to nucleic acid vaccines to rabies virus," Virology 209:569-579, 1995. By "effective amount" of a gene vaccine, we mean an amount of vaccine effective to moderate or eliminate Pseudomonas infection or Pseudomonas infection symptoms. The protein or antigen could also be used diagnostically to detect 20 antibodies in patients and, thus, predict the patient's infection status. One would preferably contact a sample obtained from an individual suspected of Pseudomonas infection with the PcrV protein or fragment thereof and detect protein/antibody binding. One would then correlate enhanced antibody binding (as compared with a control sample) with Pseudomonas aeruginosa 25 infection in the individual. One could also use the PcrV antibody or antibody fragments therapeutically. In another embodiment, the invention is the use of the antibody sequence (which we report below and in SEQ ID NOs:1-4) to produce -7recombinant single chain antibodies that may block PcrV and could also utilize the sequence in gene delivery experiments, where one would deliver eukaryotic vectors that will then lead to the production of single chain antibodies in animals for prolonged periods. The sequence could also be 5 utilized to humanize the murine monoclonal antibody to produce a product that can be utilized in human patient care. Once the antibody is safe for human use, one could: (a) administer it systemically and (b) administer it into the lungs as either a preventative treatment or as a therapy. In order to use the PcrV antibody in humans, the 10 antibody is preferably "humanized". In general, once the monoclonal antibody is obtained the heavy and light chain variable regions are cloned. These cloned fragments are then inserted into a human antibody backbone (constant regions). Thus, we can control the class of antibody (IgG, IgA, etc.) in addition to providing the binding specificity. 15 For use in the present invention, the PcrV antibody may be a monoclonal antibody or polyclonal. The antibodies may be human or humanized, particularly for therapeutic applications. Antibody fragments or derivatives, such as an Fab, F(ab') 2 or Fv, may also be used. Single-chain antibodies, for example as described in Huston, et al. (Int. Rev. Immunol. 20 10:195-217, 1993) may also find use in the methods described herein. By "effective amount" of the PcrV antibody or antibody fragment we mean an amount sufficient to moderate or eliminate Pseudomonas infection or infection symptoms. Preferably, human or humanized monoclonal or polyclonal antibodies 25 to PcrV are administered to prevent or treat infections with P. aeruginosa. In patients at high risk for P. aeruginosa infection, antibodies could be administered for prevention of infection. In addition, antibodies may be administered after the onset of infection to treat the infection. In this case, -8antibodies can be administered alone or in combination with antibiotics. Administration of antibodies in conjunction with antibiotics may allow the administration of shorter courses or lower doses of antibiotics, thereby decreasing the risk of emergence of antibiotic-resistant organisms. 57 We envision at least three types of hypothetical patients: (1) A healthy individual at risk of serious injury or bum (fire fighter, military personnel, police) would be immunized with the vaccine by a methodology (either injection or intranasal) that would give long-lived protection. A booster would be given on admission (intramuscular injection) to the hospital after injury. (2) 10 A patient who is being subjected to mechanical ventilation. (3) A patient who has been genetically diagnosed with cystic fibrosis. In addition to PcrV antibodies and antibody fragments, small molecule peptidomimetics or non-peptide mimetics can be designed to mimic the action of the PcrV antibodies in inhibiting or modulating Pseudomonas infection, 15 presumably by interfering with the action of PcrV. Methods for designing such small molecule mimics are well known (see, for example, Ripka and Rich, Cur Opin. Chem. Biol. 2:441-452, 1998; Huang, t l., Biopolymers 43:367-382, 1997; al-Obeidi, et al., Mo. Biotechnol. 9:205-223, 1998). Small molecule inhibitors that are designed based on the PcrV antibody may be 20 screened for the ability to interfere with the PcrV-PcrV antibody binding interaction. Candidate small molecules exhibiting activity in such an assay may be optimized by methods that are well known in the art, including for example, in vitro screening assays, and further refined in in vivo assays for inhibition or modulation of Pseudomonas infection by any of the methods 25 described herein or as are well known in the art. Such small molecule inhibitors of PcrV action should be useful in the present method for inhibiting or modulating a Pseudomonas infection. -9- In another aspect of the present invention, PcrV protein may be used to identify a PcrV receptor which may be present in the host cells, particularly in human cells, more particularly in human epithelial cells or macrophages. Identification of a PcrV receptor allows for the screening of small molecule 5 libraries, for example combinatorial libraries, for candidates that interfere with PcrV binding. Such molecules may also be useful in a method to inhibit or modulate a Pseudomonas infection. Our first attempts at receptor identification will be to use PcrV in pull down experiments. PcrV will be fused to glutathione S-transferase (GST) and 10 attached to column matrix for affinity chromatography of solubilized cellular extracts. Proteins binding specifically to PcrV will be eluted and subjected to amino terminal sequencing for identification. In parallel experiments PcrV will be subjected to yeast two-hybrid analysis. In this case PcrV is fused in frame with the DNA binding domain of Gal4. Once the clone is obtained it will be 15 transformed into a suitable yeast host strain. The yeast strain containing the Gal4PcrV construct will be transformed with a Hela cell cDNA bank cloned in frame with the Gal4 activation domain. Double transformants that complement the ability to utilize histidine and produce beta galactosidase (proteins that interact with PcrV) will be analyzed genetically and at the 20 nucleotide sequence level. In case the receptor is a cellular glycolipid we will utilize an overlay technique where glycolipids are separated by thin-layer chromatography and then probed with radiolabeled bacteria. The binding to specific components will be monitored by autoradiography. Similarly, epithelial and macrophage proteins will be separated by SDS-PAGE, blotted 25 onto nitrocellulose and overlaid with radiolabeled bacteria or labeled PcrV. Again, the protein components to which the bacteria bind are then identified by autoradiography. -10- Pseudomonas species are known to infect a wide spectrum of hosts within the animal kingdom and even within the plant kingdom. As will be apparent to one of ordinary skill in the art, the compositions and methods disclosed herein may have use across a wide range of organisms in inhibiting 5 or modulating diseases or conditions resulting from infection by a Pseudomonas species. The compositions and methods of the present invention are described herein particularly for application to Pseudomonas aeruginosa but it is well within the competence of one of ordinary skill in the art to apply the methods taught herein to other species. 10 EXAMPLES 1. Role of PcrV in Cytotoxicity To determine the role of PcrV in type Ill-mediated regulation/secretion, we constructed a nonpolar allele of PcrV and used the construct to replace the wild-type allele in P. aeruginosa strain PA103, a strain that is highly 15 cytotoxic in vitro (3) and causes lung epithelial damage in vivo (12, 13). Cytotoxicity and lung injury are due to the production of a specific cytotoxin, ExoU (3). PA1 03ApcrV was characterized by the expression of several extracellular products that are secreted by the P. aeruginosa type Ill system 20 which include the ExoU cytotoxin (3), PcrV (5), and a protein required for the translocation of toxins, PopD (14). SDS-polyacrylamide gel electrophoresis of concentrated culture supernatants indicated that the parental strain, PA103 is induced for production and secretion of the type IlIl proteins by growth in medium containing a chelator of calcium, nitrilotriacetic acid (NTA) (Fig. 1). 25 When an expression clone encoding PcrV was provided in trans in the parental strain, extracellular protein production in response to the presence or absence of NTA is normal. PA1 03ApcrV exhibits a calcium blind phenotype; -11extracellular protein production is strongly induced in both the presence and absence of NTA. These results suggest that the secretory system is fully functional but deregulated. This deregulated phenotype is in contrast to the calcium independent phenotype reported for an LcrV defective strain which 5 fails to produce the extracellular Yops, grows at 370C regardless of the presence or absence of calcium, and shows only partial induction of the Yops (7). Complementing PA103ApcrV with a clone expressing wild-type PcrV restored normal regulation of extracellular protein production in response to NTA induction. 10 To test the contribution of PcrV to P. aeruginosa pathogenesis, two infection models were used. In an in vitro model the parental and several mutant derivative strains were compared for their ability to cause cytotoxicity in a CHO cell infection assay (3). The negative controls in this experiment included PA103popD::0, which has been previously shown to be defective in 15 the translocation of type IlIl virulence determinants (14) and PA103AexoU, which is non-cytotoxic due to the absence of ExoU production (3, 15). After a 3 hour infection, CHO cells were unable to exclude trypan blue with the wild-type and ApcrV strain complemented with a plasmid construct expressing PcrV. Staining did not occur when CHO cells were infected with 20 the negative control strains or with PA103ApcrV (data not shown). These results suggest that PcrV expression is required for cytotoxicity. Purified recombinant PcrV was not cytotoxic when added exogenously to tissue culture cells. Since secretion of the type IlIl proteins required for translocation was unaffected by the deletion of pcrV (Fig. 1A and B), PA1 03ApcrV appears 25 to be defective in ExoU translocation. Figs. 1A and 1B are a stained gel (Fig. 1A) and Western blot (Fig. 1B) illustrating the phenotypic analysis of PA103ApcrV. The parental and ApcrV derivatives, with and without a plasmid expressing PcrV in trans, were grown -12in the absence or presence of the inducer of type Ill secretion in P. aeruginosa, nitrilotriacetic acid (NTA). The extracellular protein profile (Fig. 1A) was analyzed on a SDS-polyacrylamide gel (10%) stained with Coomassie blue. The migration of the P. aeruginosa-encoded type Ill 5 proteins is indicated to the left and the migration of molecular weight markers is indicated on the right. Fig. 1 B is a Western blot of a duplicate gel using antibodies specific for ExoU, PcrV, and PopD and 2 5 1-Protein A to detect bound IgG. Wild-type and mutant P. aeruginosa strains were tested in an acute 10 lung infection model using low and high challenge doses of bacteria. Survival measurements indicated that PcrV and PopD were required to induce a lethal infection (Fig. 2A). In experiments utilizing three independent measurements of lung injury (the flux of labeled albumin from the airspaces of the lung to the bloodstream, the flux of labeled albumin from the airspaces of the lung to the 15 pleural fluids, and the wet/dry ratio, which measures lung edema) the degree of injury caused by PA103ApcrV, the vector control strain (PA1 03ApcrVpUCP18), and PA103popD::Q were no different than the uninfected control animals (Fig. 2B). Complementation of PA1 03ApcrV with pcrV in trans restored lung injury levels to those measured with the parental 20 strain, PA1 03. Taken together these data indicate that PcrV expression is required for virulence of P. aeruginosa in the acute lung infection model and that part of the function of PcrV appears to be linked to the ability to translocate type IlIl effector proteins into eukaryotic cells. Figs. 2A and 2B are a graph (Fig. 2A) and set of bar graphs (Fig. 2B) 25 illustrating the survival and lung injury of P. aeruginosa parental and mutant strains. Referring to Fig. 2A, mice were challenged with 5 x 105 cfu of each of the indicated strains and survival was monitored for one week. Referring to Fig. 2B, lung injury was assessed by the flux of labeled albumin from the -13airspaces of the lung to the blood (lung epithelial injury), to the pleural fluid (pleural fluid) or by measuring the wet/dry ratio (lung edema). Two bacterial infectious doses were used as denoted by the solid and striped bars. Significant differences (*p<0.001) between control and test groups was 5 determined by one-way ANOVA and Dunnet multiple comparison tests. The following abbreviations were used: PA103, parental wild-type strain; ApcrV, PA103ApcrV; ApcrVpUCPpcrV, PA103ApcrV complemented with a plasmid expressing PcrV; ApcrVpUCP, PA103ApcrV with a vector control; popD::Q, PA103popD::0, a translocation defective strain. 10 2. Immunization with PcrV To determine whether immunization with PcrV protected animals from a lethal lung infection, recombinant PcrV (rPcrV) or ExoU (rExoU) were purified as histidine-tagged fusion proteins and used as antigens. Mice were immunized and subsequently challenged via their airspaces with a lethal dose 15 of strain PA103. When survival was measured, both vaccines protected the mice (Fig 3A). When lung injury was assessed, only PcrV vaccinated animals had significantly less epithelial damage and lung edema (Fig. 3B). Animals immunized with the PcrV vaccine also had significantly fewer bacteria in their lungs, suggesting that a blockade of the Pseudomonas V antigen may 20 facilitate rapid clearance of bacteria from the lung, protecting the animals from severe epithelial injury (Fig. 3B). Figs. 3A and 3B are a graph (Fig. 3A) and a set of bar graphs (Fig. 3B) illustrating the effect of immunization on survival, lung injury, and bacterial colonization. Referring to Fig. 3A, mice were immunized (PcrV, n=10; ExoU, 25 n=5; control, n=10) as indicated and challenged with strain PA103 at 5 x10 5 CFU/animal. The percent of surviving animals was determined for one week; p<0.05 by the Mantel-Cox log rank test. Referring to Fig. 3D, lung injury assessment and bacterial colonization of vaccinated animals 4 hours after -14installation of PA1 03. Lung epithelial injury, lung edema, and bacterial burden; PcrV, n=9; ExoU, n=4; and control, n=8. The final number of bacteria in the lung is indicated as the number on the Y axis x 104 CFU. Significant differences (*) for lung injury (p<0.01), lung edema (p<0.05), and bacterial 5 numbers (p<0.05) as determined by Dunnet multiple comparison test. One way ANOVA for lung injury, p=0.0005; lung edema, p=0.0437; bacterial burden, p=0.0075. To determine whether therapeutic intervention was possible, mice were passively immunized with preimmune rabbit IgG or rabbit igG specific 10 for rPcrV, rExoU, or rPopD one hour prior to airspace instillation of PA1 03 at a concentration of 5 x10 5 CFU/mouse. Antibodies to rPcrV provided complete protection to a lethal infection (Fig. 4). Anti-rExoU IgG provided partial survival, which was significantly different from the administration of control IgG, although all the surviving animals appeared severely ill during the trial. 15 Survival was not improved by the passive transfer of antibodies to another of the type Ill translocation proteins, PopD. From these results we conclude that antibodies to PcrV are highly protective in the acute lung infection model and that PcrV may be exposed on the bacterial surface or in a soluble form that is available for antibody-antigen interactions. 20 Fig. 4 is a graph of the number of animals surviving a challenge with 5 x 105 CFU/mouse of strain PA1 03. Animals were pretreated with 100 pg of immune IgG or control IgG from an unimmunized rabbit (rPcrV, preimmune serum). N=10 for each group; *p<0.05 versus control group for treatment with anti-PcrV and anti-ExoU IgG preparations by Mantel-Cox log rank test. 25 If PcrV is accessible for neutralization, then concomitant administration of the bacterial inoculum with anti-rPcrV IgG should completely protect against lung injury and lethality. IgG preparations were mixed with the inoculum (10-fold higher dose than the lethal inoculum) prior to instillation of -15the bacteria into the lung and survival was measured. Only anti-rPcrV IgG was protective against this extreme infection (Fig. 5A). Lung injury was measured in animals infected with the normal lethal dose of 5 x 10 5 bacteria. The efflux of labeled albumin from the airspaces of the lung was only 3% 5 more than uninfected controls (Fig. 5B) after co-administration of anti-rPcrV IgG. The decreased efflux of labeled protein from the lung to the pleural fluids was the same as the uninfected controls when anti-PcrV was included with the inoculum. Curiously lung edema, as measured by the wet/dry ratio, was significantly reduced by the addition of either anti-rPcrV or anti-rPopD. 10 (Fig. 5B). Thus, the concomitant administration of anti-rPcrV IgG with the bacteria was even more effective in normalizing all the lung injury parameters than vaccination. These data support the accessibility of PcrV for antibody mediated neutralization and document a clinically relevant decrease in lung injury; antibodies to PcrV may serve as therapeutic reagents in the treatment 15 of severe nosocomial pneumonia caused by Pseudomonas aeruginosa. Fig. 5 is a graph (Fig. 5A) and a set of bar graphs (Fig. 5B) illustrating survival and protection from lung injury by concomitant administration of IgG and bacterial challenge. IgG (5 pg) was mixed with either 5 x 10' (for survival assays, n=10 per group) or 5 x 10 5 (for the measurement of lung injury, n=4 to 20 6 animals per group) P. aenginosa strain PA103. This mixture was instilled into the lungs and survival (Fig. 5A) or lung injury (Fig. 5B) was-assessed. For survival, *p<0.05 versus control igG for anti-PcrV by the Mantel-Cox log rank test; for lung epithelial injury and lung edema *p<0.05 versus control igG by Dunnet multiple comparison test. One-way ANOVA for lung injury, 25 p=0.026, and lung edema, p<0.0005. In acute P. aeruginosa infections, the net effect of type ll-mediated intoxication may be to promote the dissemination of the bacterium beyond the epithelium leading to infection of the pleural fluids, spleen, liver, and -16bloodstream. Blood-bome infections with P. aeruginosa from either acute ventilator-associated pneumonia or from burn wound infections can result in a 40-80% mortality rate in spite of aggressive antibiotic treatment (16). PcrV must be a component of the type IlIl translocation complex in P. aeruginosa, 5 as mutants defective in the production of this protein are unable to intoxicate CHO cells or cause lung epithelial injury even though they are able to produce and secrete the type IlIl effectors and proteins required for translocation. Unlike PopD, which is also necessary for translocation, PcrV is accessible for antibody-mediated neutralization suggesting that antibodies 10 may be useful therapeutic agents in acute infections. 3. Methods for Examples 1 and 2 Construction of a nonpolar insertion in PcrV and complementation. A 5.0-kb EcoRl-Nsil restriction fragment encoding pcrGVHpopBD and flanking sequences was cloned into the allelic replacement vector pNOT19 (17). Two 15 Notl sites (one within pcrG and one within popB) were removed from the inserted sequences by using the Sculptor mutagenesis system (Amersham). An internal Sstl restriction fragment was deleted from pcrV, resulting in an in frame deletion of residues 17-221 (pNOTApcrV. To select for integration of the plasmid, a gene encoding tetracycline resistance (TcQ) was cloned into 20 the HindIll site of the vector (pNOT0ApcrV). The MOB cassette (17) was added as a Notl fragment. Selection of merodiploids, resolution of plasmid sequences, and confirmation of allelic replacement was performed as previously described (18). A shuttle plasmid (pUCP, 19) was used to construct a clone to complement the pcrV deletion. The coding sequence for 25 PcrV was amplified and cloned behind the control of the ExoS promoter region (20). The transcription of ExoS is coordinately regulated with the operons that control type IlIl secretion and translocation in P. aeruginosa (2). -17- The nucleotide sequence was confirmed for each DNA construct involving site specific mutagenesis, PCR amplification, or in-frame deletion. SDS-PAGE and Western blot analysis of secreted products. P. aeruginosa were grown under inducing (+NTA) or non-inducing conditions 5 (-NTA) for expression of the type Ill secreted products (18). Cultures were harvested based on optical density measurements at 540 nm and supernatant fractions were concentrated by the addition of a saturated solution of ammonium sulfate to a final concentration of 55%. Each lane of an SDS-polyacrylamide gel (11%) was loaded with 3 pl of a 20-fold 10 concentrated supernatant and stained with Coomassie blue. An identical gel was subjected to Western blot analysis as previously described (3-5) using a cocktail of rabbit antisera, which specifically recognizes ExoU, PopD, and PcrV. Protein A labeled with 1251 was used as a secondary reagent to identify bound lgG. 15 Infection models and lung injury assessments. Chinese Hamster Ovary cells (CHO) were used in an in vitro infection model designed to measure cytotoxicity and type IlIl translocation (21). Briefly, a bacterial inoculum was prepared in tissue culture medium without serum. CHO cells, which were propagated in serum containing medium, were washed and 20 infected with various P. aeruginosa strains at a multiplicity of infection of 5:1. Cultures were incubated under tissue culture conditions for 3 hours (37 0 C, 5% CO 2 ), washed, and stained with trypan blue. Permeability to the dye was determined from phase contrast photographs. Infection with the parental strain PA103, which expresses ExoU, results in trypan blue staining of 25 approximately 80% of the monolayer after 3 hours of incubation and complete destruction of the monolayer at 4-5 hours of incubation. Mouse infections and assessment of lung injury was performed as previously described (16). Briefly, male 8- to 12-week old pathogen-free BALB/c mice were purchased -18from Simonsen Laboratories (Gilroy, CA) and housed in barrier conditions. The mice were briefly anesthetized with inhaled Metofane (methoxyflurane, Pitman-Moore, Mundelein, IL) and placed supine, at an angle of approximately 30*. Fifty microliters of the bacterial inoculum was instilled 5 slowly into the left lobe using a modified 24 gauge animal feeding needle (Popper & Sons, Inc., New Hyde Park, NY) inserted into the trachea via the oropharynx. When lung injury assessments were measured, 0.5 pCi of "13 labeled human serum albumin (Merck-Frosst, Quebec, Canada), 0.05 pg of anhydrous Evans blue in ml of Ringer's lactate with 5% mouse albumin were 10 added to the instillate. After 4 hours of infection, the mice were anesthetized, blood was collected by a carotid arterial puncture and median sternotomies were performed. The lungs, pleural fluids, tracheas, oropharynxes, stomachs, and livers were harvested, and the radioactivity was measured. The percentage of radioactive albumin that left the instilled lungs and entered 15 the circulation or the pleural fluid was calculated by multiplying the counts measured in the terminal blood samples (per ml) times the blood volume (body weight X 0.07). The wet-dry ratios of the lungs were determined by adding 1 ml of water to the lungs and homogenizing the mixture. Homogenates were placed in preweighed aluminum pans and dried to 20 constant weight in an 80 0 C oven for three days. Lung homogenates were also sequentially diluted and plated on sheep blood agar for quantitative assessment of bacteria. Production of rabbit antiserum to PcrV. PooD, and ExoU. rPcrV, rPopD, and rExoU were produced as histidine tagged fusion proteins in 25 pET16b and purified by nickel chromatography as previously described (22). Rabbits were injected intradermally (10 sites) with 300 pg of recombinant protein emulsified in Freund's complete adjuvant, boosted with antigen in Freund's incomplete adjuvant, and periodically bled at 7 day intervals. For -19passive immunization, the IgG fraction was isolated using Protein A column chromatography (Pierce Chemicals, Rockford, IL). Mice were injected with 100 pg IgG (intraperitoneal injection) 1 hour before challenge with 5 x 10 5 CFU of strain PA103. For active immunization with rPcrV and rExoU, 5 endotoxin was removed from protein preparations by extraction with 1% Triton X-1 14 (23). Following the extractions, Triton X-1 14 was removed by Sephacryl S-200 chromatography. All vaccine preparations contained less than 1 ng of endotoxin per 40 pg of recombinant protein as determined by using a limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). 10 BALB/c mice were injected subcutaneously with 10 pg of recombinant proteins in Freund's complete adjuvant. At day 30 the mice were boosted with an additional 10 pg of antigen in Freund's incomplete adjuvant. On day 51 the mice were challenged by instillation of P. aeruginosa into their left lungs. 15 4. Synthesis of Monoclonal Antibodies Mice were immunized with 10 pg of purified, LPS-free, recombinant PcrV in Freund's complete adjuvant and boosted two weeks later with the same dose of antigen emulsified in Freund's incomplete adjuvant. Immunizations were performed subcutaneously. Spleens were harvested 20 from mice one week after booster doses of PcrV in Freund's incomplete adjuvant. A single spleen was placed in 5 ml of tissue culture medium without serum, cut into pieces and gently homogenized. Large pieces of tissue were allowed to settle from the homogenate and the supernatant, single-cell 25 suspension was removed and subjected to centrifugation at 1200 rpm for 10 minutes. The pelleted cells were resuspended in 10 ml of a solution to lyse red blood cells for 5 minutes and subsequently underlaid with 10 ml of fetal bovine serum. The material was centrifuged at 1200 rpm for 8 minutes, the -20supernatant was discarded and the cells were suspended in 30 ml of medium. Spleenic cells and myeloma cells (P3x63Ag8.653) were harvested by centrifugation at 1200 rpm for 10 minutes, and each pellet was separately 5 suspended in 10 ml of tissue culture medium. 108 spleen cells and 2 x 107 myeloma cells were mixed and pelleted together by centrifugation at 1200 rpm for 6 minutes. The supernatant was removed by aspiration and 1 ml of 35% polyethylene glycol (PEG) was added. The cells were suspended in this solution gently and centrifuged at 1000 rpm for 3 minutes. In some 10 experiments centrifugation was eliminated. Exactly 8 minutes after the addition of PEG, 25 ml of medium was added and the cells were gently resuspended. Following a 5 minute 1200 rpm centrifugation step, the cell pellet was suspended at a density of 1 x 106 per ml in 30% conditioned medium and 70% complete medium (with serum). 15 The cells were incubated overnight at 37 0 C. The next day the cells were harvested by centrifugation and suspended in 200 ml of 30% conditioned medium and 70% complete medium with hypoxanthine, aminopterin and thymidine (HAT). Approximately 0.2 ml of this cell suspension was added per well to ten 20 96-well plates (12 ml per 96 well plate). The density of the remaining cells was adjusted to 2.5 x 105 per ml and the cells were plated in the 96 well format. Plates were screened microscopically for single colonies and supernatants were subsequently tested for antibody production by enzyme linked immunosorbent assay using recombinant PcrV as the antigen. Clones 25 producing antibodies reactive to PcrV were subcultured to larger culture dishes and then isotyped. The binding of antibodies was tested in an enzyme linked immunosorbent assay using recombinant PcrV as the antigen (histidine -21tagged protein) coating the wells. Monoclonal antibodies were also tested in Western blot reactions using a P. aeruginosa supernatant containing native PcrV without the histidine tag. -22- 5. Identification of PcrV Antigen We obtained about three hundred cell lines producing antibodies that bound the tagged PcrV. These initial cell lines were preserved in liquid nitrogen for safekeeping. All cell lines were passaged to isolate stable 5 clones. In conjunction with isolating stable clones we developed in in vitro assay as a correlate for protection against intoxication in animal infection models. The hybridomas that were stable to passage and still produced antibodies reactive to PcrV in ELISA (approximately 80 cell lines) were 10 subsequently tested in a Fluorescence Activated Cell Sorter using the following techniques and assumptions: We reasoned that if antibody is blocking the type IlIl intoxication system, then in the presence of a monoclonal that blocks, fewer cells will be killed by our toxins. We exposed cells to each of the 80 monoclonal antibodies, added toxic bacteria, incubated, and then 15 added a dye that is only permeable to dead cell DNA (propidium iodide). Excess dye was washed away and the cells were harvested, fixed, and analyzed by FACS. Dead cells would be fluorescent since the dye leaked in and stained DNA in the nucleus. We found that if the cells were incubated with rabbit polyclonal anti 20 PcrV, mouse polyclonal anti-PcrV, or mab166 and bacteria, fewer cells died than in controls with irrelevant polyclonal antibody (anti-PopD) or the other 78 monoclonal antibodies. Mab 166 was specifically found to bind to the bacterially encoded type Ill-secreted factor termed PcrV. PcrV mediates the interaction of P. 25 aeruginosa and lung cells to facilitate the translocation of bacterial toxins that cause cellular death. This reaction is postulated to eliminate lung cells that are involved in the innate immune response to P. aeruginosa. The killing of these cells leaves the host epithelium open for P. aeruginosa colonization -23and spread to the pleural fluids and bloodstream. P. aeruginosa-encoded antibiotic resistance makes effective treatment unlikely once the bacteria have entered the bloodstream. The protection afforded by mab 166 pre- and post-bacterial instillation 5 in animal models of acute lung infection with P. aeruginosa is significant. To design antibody treatment modalities for intervention in human P. aeruginosa infections it will be necessary to produce either a human monoclonal antibody or to immunize at risk patients with the protective epitope of PcrV defined by mab 166. The goal of the work described below is to define the amino acid 10 sequence of PcrV bound by mab 166. Results We used a molecular genetic approach to define the amino acid residues bound by mab 166. PcrV possesses 294 amino acids. The approach consisted of deleting parts of the molecule at the nucleotide 15 sequence level using the polymerase chain reaction. Each product was cloned into a protein expression vector in frame with a gene encoding the glutathione S transferase protein. This strategy ensured that deletions encoding small numbers of PcrV amino acids could be detected using Western or dot blot techniques. Control bacterial lysates encoding only 20 glutathione S transferase showed no reactivity to either our anti-PcrV polyclonal or mab 166 monoclonal antibody. A total of 66 (with one full-length PcrV expression plasmid) clones were constructed, expressed, and tested for reactivity to rabbit polyclonal anti-PcrV antisera. All but one clone bound to anti-PcrV rabbit antibody 25 verifying that the expressed proteins were in-frame with PcrV. The one non reactive clone was eliminated from the analysis. None of the C-terminal deletions (n = 5 constructs) bound mab 166 suggesting that the epitope was in the C-terminal half of the protein. Only one of the N-terminal truncation -24proteins (n = 8 constructs) encoding PcrV amino acids (aa) 139-294 bound to mab 166. This experiment verified our hypothesis that the mab 166 epitope was encoded by the carboxyl terminal half of the protein. The remaining 51 constructs encoded various internal deletions of the molecule. Binding 5 analysis tabulated in Table 1, below, demonstrated that the smallest epitope recognized by mab 166 consists of aa 144-257 of PcrV. TABLE 1 PcrV Epitope Mapping All proteins are amino-terminal tagged GST-PcrV truncates. 10 Amino Acids Binding to pAb Binding to mAb 166 1-294 (full-length) yes yes (C-term truncates) 1-46 yes no 1-76 yes no 15 1-134 yes no 1-172 yes no 1-75 + 173-294 yes no (N-term truncates) 139-294 yes yes 20 148-294 yes no 159-294 yes no 164-294 yes no 194-294 yes no 261-294 yes no 25 269-294 yes no 278-294 yes no -25- Amino Acids Binding to pAb Binding to mAb 166 (internal fragments) 139-191 yes no 139-195 yes no 139-234 yes no 5 139-243 yes no 139-256 yes no 139-257 yes yes; weak 139-258 yes yes 139-259 yes yes 10 139-260 yes yes 139-261 yes yes 139-262 yes yes 139-263 yes yes 139-264 yes yes 15 139-265 yes yes 139-266 yes yes 139-274 yes yes 139-281 yes yes 140-266 yes yes 20 141-266 yes yes 142-266 yes yes 143-266 yes yes 144-266 yes yes 145-266 yes no 25 146-266 yes no 147-266 yes no 148-170 no* no 148-202 yes no 159-202 yes no 30 159-209 yes no 159-216 yes no 159-226 yes no 159-234 yes no 164-234 yes no 35 164-243 yes no 164-256 yes no 164-266 yes no 164-275 yes no 164-281 yes no 40 194-234 yes no 194-243 yes no 194-256 yes no 194-266 yes no 194-275 yes no 45 194-281 yes no -26- Amino Acids Binding to pAb Binding to mAb 166 141-258 yes yes; weak 142-258 NT** yes 143-258 yes yes 144-258 yes yes 5 141-257 yes yes 142-257 yes yes 143-257 yes yes 144-257 yes; weak yes; weak NOTES: 10 -Truncate 148-170 is the only one that is not recognized by the rabbit polyclonal control antibody. **NT; Not Tested due to an insufficient amount of bacterial lysate. -As predicted, pGEX-4T-2 vector control lysates were not recognized by either antibody. -The smallest epitope of PcrV recognizable by mAb166 appears to consist of amino acids 144-257. 15 6. Examination of PcrV-Specific Antibody Methods: Poly A+ RNA extraction: Hybridoma cell line m166 was cultured in complete Dulbeccos minimum essential medium with 4.5 g/L D-glucose, 10 mM HEPES, 50 pM 2-mercaptoethanol, 3 mM L-glutamine, and 10% heat 20 inactivated fetal calf serum, 100 U/mL penicillin and 100 pg/mL streptomycin sulfate. After the cells reached confluent state in a 75 cm 2 flask, the cells were harvested from centrifuging at 600 rpm for 5 minutes. The pellet of the cells was homogenized in 2 mL of TRIzol reagent (Life Technologies, Gaithersburg, MD), and total RNA (100 pg) was extracted after chloroform 25 fractionation, isopropanol precipitation and 70% ethanol wash. Poly A+ RNA (4 pg) was extracted with oligotex mRNA spin-column (Qiagen, Valencia, CA). RNA oligo-cappina: mRNA (250 ng) was incubated with calf intestinal phosphatase at 50*C for 1 hour to dephosphorylate non-mRNA or truncated mRNA. After the reaction, phenol/chloroform extraction and ethanol 30 precipitation was performed and the dephosphorylated RNA was incubated with tobacco acid pyrophosphatase at 37 0 C for 1 hour to remove the 5'-cap structure from full-length mRNA. After phenol/chloroform extractions and ethanol precipitation, the synthetic RNA oligo (GeneRacer RNA Oligo, Invitrogen, Carlsbad, CA) was ligated to the decapped RNA with T4 RNA -27ligase at 37*C for 1 hour. After phenol/chloroform extraction and ethanol precipitation, the RNA was suspended in 13 pL diethylpyrocarbonate-treated water. Reverse-transcribing mRNA: The RNA-oligo ligated, full-length mRNA 5 (13 pL) was reverse-transcribed with 54 base the pair primer containing a dT tail of 18 nucleotides (GeneRacer Oligo dT, Invitrogen), and Avian myeloblastosis virus reverse transcriptase at 420C for 1 hour in 20 pL reaction. After the reaction, the sample was diluted 4 times with sterile water. Amplifyinq cDNA ends by polymerase chain reaction (PCR): One 10 microliter of the cDNA was used for PCR. The 5' primer from the synthetic RNA oligo sequence (GeneRacer 5' Primer, Invitrogen) and the murine immunoglobulin gamma 2b chain CH1 region specific primer or the murine immunoglobulin kappa chain CL region specific primer were used. The cycling parameters used for the PCR reaction was; 1) 94*C, 2 minutes, 1 15 cycle, 2) 940C, 30 seconds and 72 0 C, 1 minute, 5 cycle, 3) 94"C, 30 seconds, 70 0 C, 30 seconds, and 720C, 1 minutes, 5 cycle, 4) 940C, 30 seconds, 68 0 C, 30 seconds, and 72*C, 1 minutes, 20 cycle, 5) 72*C, 10 minutes. Subcloninq and DNA sequencing: PCR products (the murine 20 immunoglobulin gamma 2b chain CH1 region derived fragment and the murine immunoglobulin kappa chain CL region derived fragment) were subcloned into the pCRIlI vector (TOPO cloning, Invitrogen) and submitted to UCSF Molecular Bioresource Center to analyze the DNA sequence. SEQ ID NO:1 is the DNA sequence of m166 heavy chain mRNA, SEQ 25 ID NO:2 is the amino acid sequence of the m166 heavy chain (IgG lilb), SEQ ID NO:3 is the DNA sequence of the m166 light chain mRNA, and SEQ ID NO:4 is the amino acid sequence of the m166 light chain. Figs. 6A, 6B and 7 examine the sequences and supply more detail. 3- Commercial Implications One could use the antibody sequence to produce recombinant single chain antibodies that may block PcrV and could also utilize the sequence in gene delivery experiments, where one would deliver eukaryotic vectors that 5 will then lead-to the production of single chain antibodies in animals for prolonged periods. Finally, the sequence could be utilized to humanize the murine monoclonal antibody to produce a product that can be utilized in human patient care. One of skill in the art would look to standard methods such as grafting the antigen binding complementarity determining regions 10 (CDRs) from variable domains of rodent antibodies on to human variable domains in order to create a humanized antibody. 7. Single Chain Antibody Against PcrV a. Assembling a single chain antibody: VH gene and VL gene were multiplied by polymerase chain reaction 15 (PCR) with specific primers for each gene. Multiplied VH and VL fragments were assembled with a linker by using PCR with primers. The assembled single chain antibody gene (scFv::m166:VH and VL genes with linker) was transferred into the cloning vector pCR4 Topo (Invitrogen, Carlsbad, CA). Then, the coding sequence of scFv::m166 was subcloned into the E. coli 20 expression vector pBAD/glli (Invitrogen) in LMG194 as the host E. coli. b. Protein induction and purification: For protein induction, in RM medium containing 0.2% glucose and 100 pg of ampicillin, the transformed E. coli was cultured overnight at 37'C in an orbital shaker (200 rpm), and the next day, 5 mL of the cultured E. coli was 25 transferred into 500 mL of the same medium and incubated for 3 hours at room temperature at 100 rpm. After L-arabinose was added at the concentration of 0.004%, the E. coli was cultured overnight. The third day, the protein was harvested from the periplasmic space of the E. coli by -29osmotic shock methods. The solution including osmotic shock derived periplasmic protein was dialyzed overnight against the lysis buffer. During the fourth day, the dialyzed solution was applied onto a nickel-NTA column to purify the hexahistidine-tagged single chain antibody. The eluted solution 5 from the nickel column was dialyzed against phosphate buffered saline overnight. On the fifth day, the dialyzed solution was applied to a centrifuge concentrator to make a higher concentration of scFv:m166. c. The binding test: The purified single chain antibody (scF::m166) was tested by using an 10 enzyme linked immunosorbent assay against recombinant PcrV and by immunoblot (western blot) against both recombinant PcrV protein and native PcrV of P. aeruginosa PA1 03. The single chain antibody will allow us to humanize the antibody utilizing phage-display techniques and to improve affinity of the antibody 15 using these techniques. The single chain antibody can be utilized as a diagnostic tool (for histology) but would not be utilized as a therapy. However, the gene for the single chain antibody can be utilized in gene therapy, so that animals would produce single-chain antibodies over an interval, which could lead to protection against P. aeruginosa infections. -30- 8. References 1. Wiener-Kronish, J.P., Sawa, T., Kurahashi, K., Ohara, M., and Gropper, M.A., "Pulmonary edema associated with bacterial pneumonia," Pulmonary Edema (eds Matthay, M.A. and ingbar, D.H.) pp. 247-267 (Marcel Dekker, Inc., New York, 1998). 2. Frank, D.W., "The exoenzyme S regulon of Pseudomonas aeruginosa," Mol Microbiol. 26:621-629 (1997). 3. Finck-Barbangon, V., et al., "ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury," Mol. Microbiol. 25:547-557 (1997). 4. Yahr, T.L., Goranson, J., and Frank, D.W., "Exoenzyme S of Pseudomonas aeruginosa is secreted by a type Ill pathway," Mol. Microbiol. 22:991-1003 (1996). 5. Yahr, T.L., Mende-Mueller, L.M., Friese, M.B., and Frank, D.W., "Identification of type III secreted products of the Pseudomonas aeruginosa exoenzyme S regulon," J Bacteriol. 179:7165-7168 (1997). 6. Hueck, C.J., "Type Ill protein secretion systems in bacterial pathogens of animals and plants," Microbiol. Mol. Biol. Rev. 62:379-433 (1998). 7. Skrzypek, E. and Straley, S.C., "Differential effects of deletion in IcrV on secretion of V antigen, regulation of the low-Ca' response, and virulence of Yersinia pestis," J. Bacteriol. 177:2530-2542 (1995). 8. Nakajima, R. and Brubaker, R.R., "Association between virulence of Yersinia pestis and suppression of gamma interferon and tumor necrosis factor alpha," Infect. Immun. 61:23-31 (1993). 9. Nakajima, R., Motin, V.L., and Brubaker, R.R., "Suppression of cytokines in mice by protein A-V antigen fusion peptide and restoration of synthesis by active immunization," Infect. Immun. 63:3021-3029 (1995). 10. Nedialkov, Y.A., Motin, V.L., and Brubaker, R.R., "Resistance to lipopolysaccharide mediated by the Yersinia pestis V antigen polyhistidine fusion peptide: amplification of interleukin-10," Infect. Immun. 63:1196-1203 (1997). 11. Nilles, M.L., Fields, K.A., and Straley, S.C., "The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG," J. Bacteriol. 180:3410-3420 (1998). 12. Kudoh, I., Wiener-Kronish, J.P., Hashimoto, S., Pittet, J.-F., and Frank, D.W., "Exoproduct secretions of Pseudomonas aeruginosa strains influence severity of alveolar epithelial injury," Am. J. Physiol. 267:L551-L556 (1994). 13. Apodaca, G., et al., "Characterization of Pseudomonas aeruginosa induced MDCK cell injury: glycosylation-defective host cells are resistant to bacterial killing," Infect. Immun. 63:1541-1551 (1995). -31- 14. Yahr, T.L., Vallis, A.J., Hancock, M.K., Barbieri, J.T., and Frank, D.W., "ExoY, a novel adenylate cyclase secreted by the Pseudomonas aeruginosa type IlIl system," Proc. Natl. Acad. Sci USA, in press (1998). 15. Finck-Barbangon, V., Yahr, T.L., and Frank, D.W., "Identification and characterization of SpcU, a chaperone required for efficient secretion of the ExoU cytotoxin,"J Bacteriol., in press (1998). 16. Sawa, T., Corry, D.B., Gropper, M.A., Ohara, M., Kurahashi, K., and Wiener-Kronish, J.P., "IL-10 improves lung injury and survival in Pseudomonas aeruginosa pneumonia," J. Immunol. 159:2858-2866 (1997). 17. Schweizer, H.P., "Allelic exchange in Pseudomonas aeruginosa using novel ColE1 -type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker," Mol. Microbiol. 6:1195-1204 (1992). 18. Frank, D.W., Nair, G., and Schweizer, H.P., "Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus," Infect. Immun. 62:554-563 (1994). 19. Schweizer, H.P., "Escherichia-Pseudomonas shuttle vectors derived from pUC18/19," Gene 97:109-112 (1991). 20. Yahr, T.L., Hovey, A.K., Kulich, S.M., and Frank, D.W., "Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene," J Bacteriol. 177:1169-1178 (1995). 21. Vallis, A.J., Yahr, T.L., Barbieri, J.T., and Frank, D.W., "Regulation of ExoS production by Pseudomonas aeruginosa in response to tissue culture conditions," Infect. Immun. submitted. 22. Yahr, T.L., Barbieri, J.T., and Frank, D.W., "Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa," J. Bacteriol. 178:1412-1419 (1996). 23. Aidi, Y. and Pabst, M.J., "Removal of endotoxin from protein solutions by phase separation using Triton X-1 14,"J Immunol. Methods 132:191-195 (1990). -32-

Claims (24)

1. An anti-PcrV monoclonal antibody or fragment thereof that recognizes an epitope in the region of amino acid residues 144 to 257 in the PcrV polypeptide amino acid sequence.

2. An anti-PcrV monoclonal antibody or fragment according to claim 1, that recognizes an epitope that includes amino acid residues 144 through 257 in the PcrV polypeptide amino acid sequence.

3. An anti-PcrV monoclonal antibody or fragment thereof according to claim 1 or claim 2 comprising the CDR's of the light chain polypeptide amino acid sequence of SEQ ID NO:4 and the CDR's of the heavy chain polypeptide amino acid sequence of SEQ ID NO: 2.

4. An anti-PcrV monoclonal antibody or fragment according to claim 3 that: (a) comprises the CDR's and the FR regions of the light chain polypeptide amino acid sequence of SEQ ID NO: 4; and (b) comprises the CDR's and the FR regions of the heavy chain polypeptide amino acid sequence of SEQ ID No:2.

5. The antibody or fragment of any of the preceding claims that is humanized or human.

6. An anti-PcrV monoclonal antibody or fragment thereof according to any one of claims 1 to 5, which is: (a) mab 166 having the amino acid sequence of the light chain of SEQ ID NO:4 and the heavy chain of SEQ ID NO: 2; (b) a humanised version of (a); or (c) a fragment of (a) or (b) able to bind to PcrV.

7. A pharmaceutical composition comprising an antibody or fragment of any of claims 1 to 6 and a pharmaceutically acceptable carrier. -33-

8. The composition according to claim 7, further comprising an antibiotic.

9. A pharmaceutical composition according to claim 7 comprising the antibody or fragment in an amount effective for treating or preventing Pseudomonas aeruginosa infection in a patient or for reducing the pathogenicity of Pseudomonas aeruginosa infection in a patient.

10. Use of an antibody or fragment according to any one of claims 1 to 6 in the manufacture of a medicament for: treating or preventing Pseudomonas aeruginosa infection in a patient; reducing the pathogenicity of Pseudomonas aeruginosa in a patient; or for modulating cytotoxicity of Pseudomonas aeruginosa to human cell(s).

11. The use according to claim 10, wherein the medicament is to be administered by inoculation.

12. The use according to claim 10, wherein the medicament is to be administered systemically.

13. The use according to claim 10, wherein the medicament is for administration to the lungs,

14. The use according to any one of claims 10 to 13, wherein the patient is a human patient.

15. A method for modulating the cytotoxicity of Pseudomonas to a human cell in vitro comprising contacting said Pseudomonas with the antibody or fragment of any of claims 1 to 6 in the presence of the human cell.

16. A nucleic acid encoding the antibody or fragment of any of claims 1 to 6. -34-

17. A method for treating or preventing Pseudomonas aeruginosa infection in a patient comprising administering to a patient an antibody or fragment according to any one of claims 1 to 6 or the composition according to claim 7 or claim 8.

18. A method for reducing the pathogenicity of Pseudomonas aeruginosa infection in a patient comprising administering to a patient an antibody or fragment according to any one of claims 1 to 6 or the composition according to claim 7 or claim 8.

19. A method for modulating the cytotoxicity of Pseudomonas to a human cell comprising administering to the cell an antibody or fragment according to any one of claims 1 to 6.

20. The method according to any one of claims 17 to 19, wherein the antibody or fragment is administered by inoculation.

21. The method according to any one of claims 17 to 19, wherein the antibody or fragment is administered systemically.

22. The method according to any one of claims 17 or 18, wherein the antibody or fragment is administered to the lungs.

23. The method according to claim 17 or 18, wherein the patient is a human patient.

24. The method according to claim 17 or 18, wherein the patient is a cystic fibrosis patient. -35-