Anti-bacterial vaccine compositions

Abstract

Gram negative bacterial virulence genes are identified, thereby allowing the identification of novel anti-bacterial agents that target these virulence genes and their products, and the provision of novel gram negative bacterial mutants useful in vaccines.

1 - 51 . (canceled)  
     
     
         52 . An attenuated Pasteurellaceae bacterium obtained by a method comprising: 
 a) mutating a Pasteurellaceae bacterium that comprises an exbB gene that encodes an amino acid sequence that is at least 70% identical to an exbB amino acid sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 141, wherein the mutating comprises performing mutagenesis to the nucleotide sequence of an exbB gene of said bacteria;    b) screening the mutated bacterium for decreased virulence compared to virulence of the unmutated bacterium; and    c) selecting as an attenuated bacterium a mutated bacterium with decreased virulence.    
     
     
         53 . The method of  claim 52 , wherein the screening comprises infecting animals and assessing animal survival, wherein attenuation correlates with increased survival.  
     
     
         54 . The bacterium of  claim 52 , wherein the mutation in said mutated bacterium results in decreased expression of a gene product encoded by the mutated gene, wherein the decreased expression of the gene product attenuates the bacterium.  
     
     
         55 . The bacterium of  claim 52 , wherein the mutation in said mutated bacterium results in expression of an inactive gene product encoded by the mutated gene.  
     
     
         56 . The bacterium of  claim 52 , wherein the mutation in said mutated bacterium results in deletion of all or part of said gene.  
     
     
         57 . The bacterium of  claim 52 , wherein the mutation in said mutated bacterium results in deletion of at least 10% of said gene.  
     
     
         58 . The bacterium of  claim 52 , wherein the mutation in said mutated bacterium results in an insertion in the gene, said insertion causing decreased expression of a gene product encoded by the mutated gene and/or expression of an inactive gene product encoded by the mutated gene.  
     
     
         59 . An attenuated Pasteurellaceae bacterium according  claim 52  selected from the group consisting of  Pasteurella haemolyrica, Pasteurella mulocida, Actinobacillus pleuropneumoniae  and  Haemophilus somnus.    
     
     
         60 . The bacterium of  claim 59  that is  Pasteurella multocida.    
     
     
         61 . A vaccine composition comprising the bacterium of  claim 52  and a pharmaceutically acceptable carrier.  
     
     
         62 . The vaccine composition of  claim 61 , further comprising an adjuvant.  
     
     
         63 . A vaccine composition comprising the bacterium of  claim 60  and a pharmaceutically acceptable carrier.  
     
     
         64 . The vaccine composition of  claim 63 , further comprising an adjuvant.  
     
     
         65 . A method for producing an attenuated Pasteurellaceae bacterium, said method comprising: 
 a) introducing a mutation in an exbB gene comprising the nucleotide sequence of SEQ ID NO. 13 or SEQ ID NO: 140 or a nucleotide sequence that is at least 70% identical to an exbB nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 140, wherein the mutation attenuates the bacterium;    b) screening the mutated bacterium for decreased virulence compared to virulence of the unmutated bacterium; and    c) selecting as an attenuated bacterium a mutated bacterium with decreased virulence.    
     
     
         66 . A purified and isolated polynucleotide selected from the group consisting of: 
 a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 140;    b) a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 141;    c) a polynucleotide that hybridizes to the complement of the polynucleotide of (a) or (b) under high stringency conditions; and    d) a polynucleotide sharing 70% or greater sequence homology to the polynucleotide of (a).    
     
     
         67 . A purified and isolated Pasteurellaceae polynucleotide, which comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 141, or a biologically or immunologically active fragment thereof, or a Pasteurellaceae species homolog thereof sharing 70% or greater sequence homology to the amino acid sequence of SEQ ID NO:  14  or SEQ ID NO: 141.  
     
     
         68 . The polynucleotide of  claim 66 , which comprises a nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 140, or a fragment thereof, or a Pasteurellaceae species homolog thereof sharing 70% or greater sequence homology to the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 140.  
     
     
         69 . The polynucleotide of  claim 66 , which is a DNA.  
     
     
         70 . A vector comprising the DNA of  claim 69 .  
     
     
         71 . The vector of  claim 70  that is an expression vector, wherein the DNA is operatively linked to an expression control DNA sequence.  
     
     
         72 . A host cell stably transformed or transfected with the DNA of  claim 69 , in a manner allowing the expression of the encoded polypeptide in the host cell.  
     
     
         73 . A method for producing a recombinant polypeptide comprising culturing the host cell of  claim 72  in a nutrient medium and isolating the encoded polypeptide from the host cell or the nutrient medium.  
     
     
         74 . A purified polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 141, or a fragment thereof, or a Pasteurellaceae species homolog thereof sharing 70% or greater sequence homology to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 141.  
     
     
         75 . An antibody that is specifically reactive with a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 141, or a fragment thereof, or a Pasteurellaceae species homolog thereof sharing 70% or greater sequence homology to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 141.  
     
     
         76 . The antibody of  claim 75 , that is a monoclonal antibody.