Brucella melitensis mutants and methods

Abstract

Certain attenuated mutants of Brucella , especially B. melitensis, B. abortus, B. suis and B. ovis , when administered to a human or animal trigger a protective immune response such that subsequent challenge with virulent Brucella of the same species does not result in disease or results in much less severe symptoms. Functional inactivation of galE, a virB gene or the operon (ORFs 1087-1090) comprising the gene encoding β-hexosaminidase (BMEI1087) and a lytic murein transglycosylase gene (BMEI1088). A specific example of the attenuated galE mutant which produces a protective immune response is B. melitensis GR024. The specific example of an inactivated ORF1087-1090 operon is B. melitensis GR026; it has an insertion mutation in the promoter region upstream of ORF 1090. Vaccination with live cells of either or both of these mutants results in a T cell response which protects the human or animal against challenge with virulent B. melitensis . Similar strategies for protective immunity using live attenuated mutants are useful for B. abortus, B. suis and B. ovis as well.

1 . An attenuated strain of  Brucella  in which there is a mutation which functionally inactivates or prevents expression of at least one of the galE and having at least 85% nucleotide sequence identity to SEQ ID NO:28, the gene encoding lytic murein transglycosylase and having at least 85% nucleotide sequence identity to nucleotides 7908-10817 of SEQ ID NO:26, β-hexosaminidase and having at least 85% nucleotide sequence identity with nucleotides 6688-7740 of SEQ ID NO:26, or a gene encoding deoxyguanosinetriphosphate triphosphohydrolase and having at least 85% nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27.  
     
     
         2 . The attenuated strain of  claim 1 , wherein said mutation is in a  Brucella melitensis  16M genetic background and wherein said a mutation which functionally inactivates the galE gene of BMEI1090, lytic murein transglycosylase gene of BME11088 and β-hexosaminidase of BMEI1087 or a gene encoding deoxyguanosinetriphosphate triphosphohydrolase and having at least 85% nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27.  
     
     
         3 . The attenuated strain of  claim 1 , wherein said strain is a  Brucella melitensis  in which there is a mutation which functionally inactivates ORF BME10971 or its counterpart in a species of  Brucella  other than  B. melitensis , the ORF BME11090 or its counterpart in a species of  Brucella  other than  B. melitensis , and the operon comprising ORFs BME11090, 1089, 1088 and 1087 or a counterpart operon in a species of  Brucella  other than  B. melitensis.    
     
     
         4 . The attenuated strain of  Brucella  of  claim 1 , wherein said mutation is a polar insertion mutation.  
     
     
         5 . The attenuated strain of  Brucella  of  claim 1 , wherein said  Brucella  is  Brucella melitensis.    
     
     
         6 . The attenuated mutant strain of  Brucella  of  claim 1 , wherein said  Brucella  is  Brucella abortus.    
     
     
         7 . The attenuated strain of  Brucella melitensis  of  claim 3 , wherein said attenuated mutant is GRO24 or GRO26.  
     
     
         8 . The attenuated strain of  Brucella  of  claim 1  in which there is a deletion in the gene encoding deoxyguanosine triphosphate hydrolase.  
     
     
         9 . The attenuated strain of  claim 1 , wherein said mutant strain expresses a listeriolysin O gene from  Listeria monocytogenes.    
     
     
         10 . An immunogenic composition comprising live cells of at least one attenuated mutant strain of the  Brucella  of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         11 . The immunogenic composition of  claim 10 , wherein said attenuated mutant strain of  Brucella  is a  Brucella melitensis.    
     
     
         12 . The immunogenic composition of  claim 11 , wherein said attenuated mutant is GR024 or GR026.  
     
     
         13 . The immunogenic composition of  claim 10  wherein said  Brucella  is  Brucella abortus.    
     
     
         14 . A method of protecting a human or animal against infection by administering an effective amount of the immunogenic composition of  claim 10 .  
     
     
         15 . The method of  claim 14 , wherein said immunogenic composition comprises at least one attenuated mutant strain of  Brucella melitensis.    
     
     
         16 . The method of  claim 13 , wherein said attenuated mutant strain is GR024 or GR026.  
     
     
         17 . The method of  claim 14 , wherein said immunogenic composition comprises at least one attenuated mutant strain of  Brucella abortus.    
     
     
         18 . A method of identifying epitopic peptides of  B. melitensis  comprising the steps of: 
 (a) infecting macrophage cells in culture with  B. melitensis;      (b) culturing the macrophage cells infected with  B. melitensis;      (c) collecting MHC class I-peptide complexes from the cells cultured in step (b);    (d) eluting the peptides from the collected complexes of step (c); and    (e) characterizing the peptides eluted in step (d).    
     
     
         19 . An epitopic peptide identified by the method of  claim 18 , wherein said peptide is derived from the HdeA protein of  B. melitensis.