Recombinant BCG strains with enhanced ability to escape the endosome

Abstract

Mycobacterium strains that have an enhanced ability to elicit a Histocompatibility-Class I-restricted CD8 + T cell immune response are provided. The Mycobacterium strains are genetically engineered to express a endosomalytic protein that is active at neutral pH (e.g. Perfringolysin O), permitting escape of the Mycobacterium from endosomes into the cytoplasm of the cell. The invention also provides vaccine preparations containing the Mycobacterium strains.

1 . A  Mycobacterium  that is genetically engineered to include an expressable and secretable functional endosomalytic protein that is active at a pH of 6-8. 
     
     
         2 . The  Mycobacterium  of  claim 1  wherein said expressable and secretable functional endosomalytic protein is perfringolysin or a functional variant thereof. 
     
     
         3 . The  Mycobacterium  of  claim 1  wherein an amino acid sequence of said expressable and secretable functional endosomalytic protein is represented by SEQ ID NO. 2. 
     
     
         4 . The  Mycobacterium  of  claim 1  wherein said expressable and secretable functional endosomalytic protein is encoded by a gene sequence specific for perfringolysin or a mutant thereof. 
     
     
         5 . The  Mycobacterium  of  claim 4  wherein said, gene sequence is selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3. 
     
     
         6 . The  Mycobacterium  of  claim 1 , wherein said mycobacterium is genetically engineered to express an apoptotic protein or a functional enhancer of apoptosis. 
     
     
         7 . The  Mycobacterium  of  claim 6 , wherein said apoptotic protein or said functional enhancer of apoptosis is selected from the group consisting of caspase 8, death-receptor-5, Fas and Fas cytoplasmic domain/CD4 ectodomain fusion protein. 
     
     
         8 . The  Mycobacterium  of  claim 1 , wherein said mycobacterium is genetically engineered to functionally express a gene of interest. 
     
     
         9 . The  Mycobacterium  of  claim 1 , wherein said mycobacterium is genetically engineered to functionally express
 an apoptotic protein or a functional enhancer of apoptosis; and   a gene of interest.   
     
     
         10 . A  Mycobacterium  which is genetically engineered to include an expressable and secretable functional endosomalytic protein that is active at a pH present in endosomes of cells infected by said mycobacterium. 
     
     
         11 . The  Mycobacterium  of  claim 10  wherein said expressable and secretable functional endosomalytic protein is perfringolysin or a functional variant thereof. 
     
     
         12 . The  Mycobacterium  of  claim 10  wherein an amino acid sequence of said expressable and secretable functional endosomalytic protein is represented by SEQ ID NO. 2. 
     
     
         13 . The  Mycobacterium  of  claim 10  wherein said expressable and secretable functional endosomalytic protein is encoded by a gene sequence specific for perfringolysin or a mutant thereof. 
     
     
         14 . The  Mycobacterium  of  claim 13  wherein said gene sequence is selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 3. 
     
     
         15 . The  Mycobacterium  of  claim 10  wherein said mycobacterium is BCG. 
     
     
         16 . The  Mycobacterium  of  claim 10 , wherein said mycobacterium is genetically engineered to express an apoptotic protein or a functional enhancer of apoptosis. 
     
     
         17 . The  Mycobacterium  of  claim 16 , wherein said apoptotic protein or said functional enhancer of apoptosis is selected from the group consisting of caspase 8, death-receptor-5, Fas and Fas cytoplasmic domain/CD4 ectodomain fusion protein. 
     
     
         18 . The  Mycobacterium  of  claim 10 , wherein said mycobacterium is genetically engineered to functionally express a gene of interest. 
     
     
         19 . The  Mycobacterium  of  claim 10 , wherein said mycobacterium is genetically engineered to functionally express
 an apoptotic protein or a functional enhancer of apoptosis; and   a gene of interest.   
     
     
         20 . A method of enabling a  Mycobacterium  to escape from endosomes, comprising the step of
 genetically engineering said  Mycobacterium  to contain, express and secrete a functional endosomalytic protein.   
     
     
         21 . The method of  claim 20 , wherein said functional endosomalytic protein is perfringolysin O or a mutant thereof. 
     
     
         22 . The method of  claim 21 , wherein said functional endosomalytic protein is a mutant perfringolysin O encoded by SEQ ID NO: 3. 
     
     
         23 . The method of  claim 20 , wherein said  Mycobacterium  is an attenuated  Mycobacterium.    
     
     
         24 . The method of  claim 23 , wherein said attenuated  Mycobacterium  is BCG. 
     
     
         25 . The method of  claim 20 , wherein said  Mycobacterium  is genetically engineered to express an apoptotic protein or a functional enhancer of apoptosis. 
     
     
         26 . The method of  claim 25 , wherein said apoptotic protein or said functional enhancer of apoptosis is selected from the group consisting of caspase 8, death-receptor-5, Fas and Fas cytoplasmic domain/CD4 ectodomain fusion protein. 
     
     
         27 . The method of  claim 20 , wherein said  Mycobacterium  is genetically engineered to functionally express a gene of interest. 
     
     
         28 . The method of  claim 20 , wherein said  Mycobacterium  is genetically engineered to functionally express van apoptotic protein or a functional enhancer of apoptosis; and
 a gene of interest.   
     
     
         29 . A vaccine preparation, comprising
 a  Mycobacterium  genetically engineered to express and secrete a functional endosomalytic protein, wherein said functional endosomalytic protein is active at neutral pH.   
     
     
         30 . The vaccine preparation of  claim 29 , wherein said functional endosomalytic protein is perfringolysin O. 
     
     
         31 . The vaccine preparation of  claim 30 , wherein said functional endosomalytic protein is a mutant perfringolysin O encoded by SEQ ID NO: 3. 
     
     
         32 . The vaccine preparation of  claim 29 , wherein expression of said functional endosomalytic protein expressed by said  Mycobacterium  permits escape of said recombinant  Mycobacterium  from endosomes. 
     
     
         33 . The vaccine preparation of  claim 29 , wherein said  Mycobacterium  is an attenuated  Mycobacterium.    
     
     
         34 . The vaccine preparation of  claim 33 , wherein said attenuated  Mycobacterium  is BCG. 
     
     
         35 . The vaccine preparation of  claim 29 , wherein said  Mycobacterium  is genetically engineered to express an apoptotic protein or a functional enhancer of apoptosis. 
     
     
         36 . The vaccine preparation of  claim 35 , wherein said apoptotic protein or said functional enhancer of apoptosis is selected from the group consisting of caspase 8, death-receptor-5, Fas and Fas cytoplasmic domain/CD4 ectodomain fusion protein. 
     
     
         37 . The vaccine preparation of  claim 29 , wherein said  Mycobacterium  is genetically engineered to functionally express a gene of interest. 
     
     
         38 . The vaccine preparation of  claim 29 , wherein said  Mycobacterium  is genetically engineered to functionally express
 an apoptotic protein or a functional enhancer of apoptosis; and   a gene of interest.