Active immunization using a siderophore receptor protein

Abstract

The invention provides a vaccine for immunizing poultry and other animals against infection by a gram-negative bacteria, and a method of immunizing an animal using the vaccine The vaccine may contain purified siderophore receptor proteins derived from a single strain or species of gram-negative bacteria or other organism, which are cross-reactive with siderophores produced by two or more strains, species or genera of gram-negative bacteria. The invention further provides a process for isolating and purifying the siderophore receptor proteins, and for preparing a vaccine containing the proteins. Also provided is a method for diagnosing gram-negative sepsis.

What is claimed is:  
     
         1 . A vaccine for use in immunizing an animal against an infection by a gram-negative bacteria, comprising: 
 a siderophore receptor protein derived from the outer membrane of a first gram-negative bacteria, in combination with a physiologically-acceptable carrier;    the siderophore receptor protein effective to stimulate production of an antibody immunoreactive with the first gram-negative bacteria, and with a second gram-negative bacteria that expresses a siderophore receptor protein and is of a different strain or species than the first gram-negative bacteria.    
     
     
         2 . The vaccine according to  claim 1 , comprising 1-15 siderophore receptor proteins having different molecular masses derived from the first gram-negative bacteria.  
     
     
         3 . The vaccine according to  claim 1 , comprising a siderophore receptor protein selected from the group consisting of aerobactin receptor protein, coprogen receptor protein, enterochelin receptor protein, citrate receptor protein, multocidin receptor protein, and ferrichrome receptor protein.  
     
     
         4 . The vaccine according to  claim 1 , wherein the first gram-negative bacteria is selected from the group consisting of  Escherichia coli , Salmonella spp., Pasteurella spp., Klebsiella spp., and Pseudomonas spp.  
     
     
         5 . The vaccine according to  claim 4 , wherein the first gram-negative bacteria is selected from the group consisting of  Escherichia coli, Salmonella agona, Salmonella blockley, Salmonella enteriditis, Salmonella hadar, Salmonella heidelberg, Salmonella Montevideo, Salmonella senftenberg, Salmonella cholerasuis, Salmonella typhimurium, Pasteurella multocida, Klebsiella pneumoniae, and Pseudomonas aeruginosa.    
     
     
         6 . The vaccine according to  claim 4 , wherein the first gram-negative bacteria is  Escherichia coli , and the second gram-negative bacteria is selected from the group consisting of Salmonella spp.,  Pseudomonas aeruginosa, Klebsiella pneumoniae , and  Pasteurella multocida.    
     
     
         7 . The vaccine according to  claim 4 , wherein the first gram-negative bacteria is  Escherichia coli  serotype 078.  
     
     
         8 . The vaccine according to  claim 7 , wherein the siderophore receptor protein derived from  Escherichia coli  has a molecular weight of about 91-92 kDa, 89 kDa, 84 kDa, 78 kDa, 74 kDa, or 72 kDa, as determined by SDS-PAGE.  
     
     
         9 . The vaccine according to  claim 1 , wherein the first gram-negative bacteria is a species of Pasteurella, and the second gram-negative bacteria is selected from the group consisting of Salmonella spp. and  Eschericnia coli.    
     
     
         10 . The vaccine according to  claim 9 , wherein the siderophore receptor protein derived from Pasteurella spp. has a molecular weight of about 96 kDa, 84 kDa, or 80 kDa, as determined by SDS-PAGE.  
     
     
         11 . The vaccine according to  claim 1 , wherein the first gram-negative bacteria is a species of Salmonella, and the second gram-negative bacteria selected from the group consisting of  Escherichia coli , Pseudomonas, Klebsiella, and  Pasteurella multocida.    
     
     
         12 . The vaccine according to  claim 10 , wherein the siderophore receptor protein derived from Salmonella spp. has a molecular weight of about 89 kDa, 81 kDa, or 72 kDa, as determined by SDS-PAGE.  
     
     
         13 . The vaccine according to  claim 1 , wherein the vaccine further comprises a non-iron-regulated outer membrane protein derived from  Escherichia coli , having a molecular weight of about 34-38 kDa; 
 the protein being reactive with lactoferrin, ferrichrome, or both; and capable of inducing an antibody that is cross-reactive with an outer membrane protein of the group consisting of  Escherichia coli , Salmonella spp., Pseudomonas spp., Klebsiella spp., and Pasteurella spp.    
     
     
         14 . The vaccine according co  claim 1 , wherein the vaccine further comprises 1-15 siderophore receptor proteins derived from a gram-negative bacteria of a different strain or species than the first gram-negative bacteria.  
     
     
         15 . The vaccine according to  claim 1 , wherein the vaccine comprises the siderophore receptor protein in an amount and type effective to immunize the animal against an infection by the first gram-negative bacteria.  
     
     
         16 . The vaccine according to  claim 15 , wherein the vaccine comprises the siderophore receptor protein in an amount and type effective to immunize the animal against an infection by two or more strains or species of gram-negative bacteria that express a siderophore receptor protein.  
     
     
         17 . The vaccine according to  claim 15 , effective to stimulate production of antibody to the siderophore receptor protein in an adult animal and subsequent transfer of said antibody to progeny of the adult.  
     
     
         18 . The vaccine according to  claim 1 , wherein the carrier is a liquid, and the amount of the siderophore receptor protein in the vaccine is about 25-5000 μg/ml.  
     
     
         19 . The vaccine according to  claim 18 , wherein the carrier is selected from the group consisting of physiological saline, pohosphate-buffered saline, Tris(hydroxymethyl aminomethane), and Tris-buffered saline.  
     
     
         20 . The vaccine according to  claim 1 , wherein the carrier is in the form of a solution, water-in-oil emulsion, liposomes, or a metabolizable solid matrix.  
     
     
         21 . The vaccine according to  claim 1 , further comprising an adjuvant selected from the group consisting of aluminum hydroxide, aluminum phosphate, and Freund's Incomplete Adjuvant.  
     
     
         22 . A process for preparing a substantially pure siderophore receptor protein from gram-negative bacteria, comprising: 
 (a) contacting the bacteria with a composition consisting essentially of sodium dodecyl sulfate to extract the siderophore receptor protein from the outer membrane of the bacteria;    (b) separating the siderophore receptor protein from the composition to form a protein fraction; and    (c) removing the sodium dodecyl sulfate from the protein fraction to provide a substantially pure siderophore receptor protein.    
     
     
         23 . The process according to  claim 22 , wherein the sodium dodecyl sulfate composition is a buffer solution containing about 0.1-4% sodium dodecyl sulfate (0.2%).  
     
     
         24 . The process according to  claim 22 , wherein contacting step (a) is under non-reducing conditions.  
     
     
         25 . The process according to  claim 22 , wherein separating step (b) is by a chromatographic process selected from the group consisting of affinity chromatograph, ion exchange chromatography, and size exclusion chromatography.  
     
     
         26 . The process according to  claim 25 , wherein step (b) comprising separating the siderophore receptor proteins on a 12.5% acrylamide reducing gel.  
     
     
         27 . The process according to  claim 22 , further comprising prior to step (a), the step of culturing the gram-negative bacteria in a substantially iron-free media to stimulate production of the siderophore receptor protein.  
     
     
         28 . A method for making a vaccine containing a siderophore receptor protein, comprising: 
 (a) culturing a gram-negative bacteria in a substantially iron-free media to stimulate production of a siderophore receptor protein by the gram-negative bacteria;    (b) isolating the siderophore receptor protein from the bacteria to provide a substantially pure siderophore receptor protein; and    (c) combining the siderophore receptor protein with a physiologically-acceptable carrier to form a vaccine effective for immunizing an animal against infection by two or more strains or species of gram-negative bacteria.    
     
     
         29 . The method according to  claim 28 , wherein the siderophore receptor protein is isolated from outer membranes of the bacteria using sodium dodecyl sulfate under non-reducing conditions.  
     
     
         30 . A method for immunizing an animal against an infection by a gram-negative bacteria, comprising: 
 administering to the animal, an effective amount of a composition comprising a siderophore receptor protein derived from the outer membrane of a first gram-negative bacteria, in combination with a physiologically-acceptable carrier;    the siderophore receptor protein effective to stimulate production of an antibody immunoreactive with the first gram-negative bacteria, and with a second gram-negative bacteria that expresses a siderophore receptor protein and is of a different strain or species than the first gram-negative bacteria.    
     
     
         31 . The method according to  claim 30 , wherein the vaccine is administered by subcutaneous injection, intramuscular injection, sustained release repository, aerosolization, or inoculation into an egg.  
     
     
         32 . A method of diagnosing an infection of a gram-negative bacteria in an animal, comprising: 
 (a) obtaining a body material from the animal;    (b) immobilizing the body material on a substrate;    (c) contacting the immobilized material with an antibody immunoreactive with a siderophore receptor protein to form an immobilized complex of the antibody and the siderophore receptor protein;    (d) contacting the immobilized antibody/protein complex with a labelled anti-species antibody reactive with said protein-reactive antibody; and    (e) detecting the labelled antibody attached to the immobilized complex.    
     
     
         33 . A serum suitable for treatment of an animal infected with a gram-negative bacteria, comprising: 
 a semi-purified blood serum of an animal inoculated with a siderophore receptor protein derived from a first gram-negative bacteria, containing antibodies reactive with the first gram-negative bacteria and with a second gram-negative bacteria that expresses a siderophore receptor protein and is of a strain or species different than the first gram-negative bacteria.    
     
     
         34 . The serum according to  claim 33 , wherein the siderophore receptor protein is derived from cells of the first gram-negative bacteria cultured in a medium substantially replete of ferric iron.  
     
     
         35 . A method for immunizing an animal, the method comprising administering to an animal a composition comprising: 
 siderophore receptor proteins (SRPs) extracted from an outer membrane of a bacterium of family Enterobacteriaceae, family Pasteurellaceae, or family Pseudomonadaceae, wherein the SRPs comprise molecular weights of about 72-96 kDa;    a non-iron regulated protein obtained from an outer membrane of a bacterium of family Enterobacteriaceae or family Pasteurellaceae, wherein the non-iron regulated protein comprises a molecular weight of about 34-38 kDa.; and    a physiologically acceptable carrier.    
     
     
         36 . The method of  claim 35  wherein the bacterium of family Enterobacteriaceae is  Escherichia coli , and the composition comprises at least 2 SRPs.  
     
     
         37 . The method of  claim 35  wherein the bacterium of family Enterobacteriaceae is Salmonella spp., and the composition comprises at least 3 SRPs.  
     
     
         38 . The method of  claim 37  wherein the Salmonella spp. is serotype B, serotype C 1 , serotype C 2 , serotype D 1 , or serotype E 3 .  
     
     
         39 . The method of  claim 35  wherein the bacterium of family Pasteurellaceae is Pasteurella spp.  
     
     
         40 . The method of  claim 35  wherein the Pasteurella spp. is  Pasteurella multocida , and the composition comprises at least 3 SRPs.  
     
     
         41 . A method for immunizing an animal, the method comprising administering to an animal a composition comprising: 
 SRPs extracted from an outer membrane of a bacterium of family Enterobacteriaceae, wherein the SRPs comprise molecular weights of about 72-96 kDa;    a non-iron regulated protein obtained from an outer membrane of a bacterium of family Enterobacteriaceae, wherein the non-iron regulated protein comprises a molecular weight of about 34-38 kDa; and    a physiologically acceptable carrier; wherein a Pasturella spp. infection in the animal is prevented or eliminated.    
     
     
         42 . The method of  claim 41  wherein the bacterium of family Enterobacteriaceae is  Escherichia coli , and the composition comprises at least 2 SRPs.  
     
     
         43 . The method of  claim 41  wherein the bacterium of family Enterobacteriaceae is Salmonella spp., and the composition comprises at least 3 SRPs.  
     
     
         44 . The method of  claim 43  wherein the Salmonella spp. is serotype B, serotype C 1 , C 2 , D 1 , or E 3 .  
     
     
         45 . The method of  claim 41  wherein the bacterium of family Pasteurellaceae is Pasteurella spp.  
     
     
         46 . The method of  claim 41  wherein the Pasteurella spp. is  Pasteurella multocida , and the composition comprises at least 3 SRPs.  
     
     
         47 . A method for immunizing an animal, the method comprising administering to an animal a composition comprising: 
 at least 2 SRPs extracted from an outer membrane of an  Escherichia coli , wherein the SRPs comprise molecular weights of about 72-96 kDa;    a non-iron regulated protein obtained from an outer membrane of a bacterium of family Enterobacteriaceae, wherein the non-iron regulated protein comprises a molecular weight of about 34-38 kDa; and    a physiologically acceptable carrier; wherein a Salmonella spp. infection in the animal is prevented or eliminated.    
     
     
         48 . A method for immunizing an animal, the method comprising administering to an animal a composition comprising: 
 at least 3 SRPs extracted from an outer membrane of a first Salmonella spp., wherein the SRPs comprise molecular weights of about 72-96 kDa;    a non-iron regulated protein obtained from an outer membrane of a bacterium of family Enterobacteriaceae, wherein the non-iron regulated protein comprises a molecular weight of about 34-38 kDa; and    a physiologically acceptable carrier; wherein a Salmonella spp. infection in the animal caused by a second Salmonella spp. is prevented or eliminated, wherein the first Salmonella spp. and the second Salmonella spp. are the same species.    
     
     
         49 . A method for immunizing an animal, the method comprising administering to an animal a composition comprising: 
 at least 3 SRPs extracted from an outer membrane of a first Salmonella spp., wherein the SRPs comprise molecular weights of about 72-96 kDa;    a non-iron regulated protein obtained from an outer membrane of a bacterium of family Enterobacteriaceae, wherein the non-iron regulated protein comprises a molecular weight of about 34-38 kDa; and    a physiologically acceptable carrier; wherein a Salmonella spp. infection in the animal caused by a second Salmonella spp. is prevented or eliminated, wherein the first Salmonella spp. and the second Salmonella spp. are different species.    
     
     
         50 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a body sample from an animal;    providing an antibody that binds to an SRP of a bacterium of family Enterobacteriaceae, family Pasteurellaceae, or family Pseudomonadaceae;    contacting the body sample with an antibody to form a complex; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a gram-negative infection.    
     
     
         51 . The method of  claim 50  wherein the body sample is immobilized on a substrate.  
     
     
         52 . The method of  claim 50  wherein the Enterobacteriaceae is  Escherichia coli  or Salmonella spp.  
     
     
         53 . The method of  claim 50  wherein the Pasteurellaceae is Pasteurella spp.  
     
     
         54 . The method of  claim 50  wherein the gram-negative infection is caused by  Escherichia coli , Salmonella spp., Pasteurella spp., or Pseudomonadaceae spp.  
     
     
         55 . The method of  claim 50  wherein the antibody is a first antibody, wherein the detecting comprises contacting the complex with a second antibody reactive with the first antibody.  
     
     
         56 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a body sample from an animal;    providing an antibody that binds to an SRP of a bacterium of family Enterobacteriaceae;    contacting the body sample with an antibody to form a complex; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a Pasturella spp. infection.    
     
     
         57 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a body sample from an animal;    providing an antibody that binds to an SRP of a bacterium of family Pasteurellaceae;    contacting the body sample with an antibody to form a complex; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a  Escherichia coli  or Salmonella spp. infection.    
     
     
         58 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a body sample from an animal;    providing an antibody that binds to an SRP of an  Escherichia coli;      contacting the body sample with an antibody to form a complex; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a Salmonella spp. infection.    
     
     
         59 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a body sample from an animal;    providing an antibody that binds to an SRP of a Salmonella spp.;    contacting the body sample with an antibody to form a complex; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has an  Eschericha coli  infection.    
     
     
         60 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a substrate comprising an immobilized SRP, the SRP extracted from an outer membrane of a bacterium of family Enterobacteriaceae, family Pasteurellaceae, or family Pseudomonadaceae;    providing serum comprising an antibody obtained from an animal;    contacting the serum with the immobilized SRP to form a complex comprising the immobilized SRP and the antibody; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a gram-negative infection.    
     
     
         61 . The method of  claim 60  wherein the Enterobacteriaceae is  Escherichia coli  or Salmonella spp.  
     
     
         62 . The method of  claim 60  wherein the Pasteurellaceae is Pasteurella spp.  
     
     
         63 . The method of  claim 60  wherein the gram-negative infection is caused by  Escherichia coli , Salmonella spp., Pasteurella spp., or Pseudomonas spp.  
     
     
         64 . The method of  claim 60  wherein the antibody is a first antibody, wherein the detecting comprises contacting the complex with a second antibody reactive with the first antibody.  
     
     
         65 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a substrate comprising an immobilized SRP, the SRP extracted from an outer membrane of a bacterium of family Enterobacteriaceae;    providing serum comprising an antibody obtained from an animal;    contacting the serum with the immobilized SRP to form a complex comprising the immobilized SRP and the antibody; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a Pasturella spp. infection.    
     
     
         66 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a substrate comprising an immobilized SRP, the SRP extracted from an outer membrane of a bacterium of family Pasteurellaceae;    providing serum comprising an antibody obtained from an animal;    contacting the serum with the immobilized SRP to form a complex comprising the immobilized SRP and the antibody; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a  Escherichia coli  or Salmonella spp. infection.    
     
     
         67 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a substrate comprising an immobilized SRP, the SRP extracted from an outer membrane of an  Escherichia coli;      providing serum comprising an antibody obtained from an animal;    contacting the serum with the immobilized SRP to form a complex comprising the immobilized SRP and the antibody; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has a Salmonella spp. infection.    
     
     
         68 . A method for diagnosing an infection by a gram-negative bacteria in an animal, the method comprising: 
 providing a substrate comprising an immobilized SRP, the SRP extracted from an outer membrane of a bacterium of Salmonella spp.;    providing serum comprising an antibody obtained from an animal;    contacting the serum with the immobilized SRP to form a complex comprising the immobilized SRP and the antibody; and    detecting the complex, wherein the presence of a detectable complex indicates the animal has an  Escherichia coli  infection.    
     
     
         69 . A method for immunizing an animal comprising administering to an animal an antibody, wherein the antibody binds to an SRP obtained from an outer membrane of a bacterium of family Enterobacteriaceae, family Pasteurellaceae, or family Pseudomonadaceae, wherein the antibody prevents or eliminates infection by a heterologous bacterium.  
     
     
         70 . The method of  claim 69  wherein the bacterium of family Enterobacteriaceae is  Escherichia coli , and the composition comprises at least 2 SRPs.  
     
     
         71 . The method of  claim 69  wherein the bacterium of family Enterobacteriaceae is Salmonella spp., and the composition comprises at least 3 SRPs.  
     
     
         72 . The method of  claim 71  wherein the Salmonella spp. is serotype B, serotype C 1 , serotype C 2 , serotype D 1 , or serotype E 3 .  
     
     
         73 . The method of  claim 69  wherein the bacterium of family Pasteurellaceae is Pasteurella spp.  
     
     
         74 . The method of  claim 69  wherein the Pasteurella spp. is  Pasteurella multocida , and the composition comprises at least 3 SRPs.  
     
     
         75 . The method of  claim 69  wherein the bacterium is family Enterobacteriaceae and the heterologous bacterium is family Pasteurellaceae.  
     
     
         76 . The method of  claim 69  wherein the bacterium is family Pasteurellaceae and the heterologous bacterium is family Enterobacteriaceae.  
     
     
         77 . A method for decreasing intestinal colonization by an Enterobacteriaceae, the method comprising: 
 administering to an animal a vaccine composition comprising SRPs extracted from an outer membrane of a first bacterium of family Enterobacteriaceae and a physiologically acceptable carrier, wherein the SRPs comprise molecular weights of about 72-96 kDa, and wherein the presence of a second bacterium of family Enterobacteriaceae in the animal's intestine is decreased after administration.    
     
     
         78 . The method of  claim 77  wherein the SRPs are extracted from  Escherichia coli  or Salmonella spp.  
     
     
         79 . The method of  claim 77  wherein the genus of the first bacterium and the genus of the second bacterium are the same.  
     
     
         80 . The method of  claim 77  wherein the genus of the first bacterium and the genus of the second bacterium are different.  
     
     
         81 . A method for decreasing intestinal colonization by Salmonella spp., the method comprising: 
 administering to an animal a vaccine composition comprising SRPs obtained from an outer membrane of first Salmonella spp. and a physiologically acceptable carrier, wherein the presence of a second Salmonella spp. in the animal's intestine is decreased compared to an animal that was not administered the vaccine composition.