Hyperblebbing shigella strains

Abstract

Hyperblebbing Shigella strains are generated by disrupting one or more components of the Tol-Pal system. The blebs from these strains are useful immunogens for vaccination. The individual proteins found in these blebs can also be used as immunogens.

1 . A  Shigella  bacterium which expresses no more than 4 of TolA, TolB, TolQ, TolR and Pal proteins. 
     
     
         2 . The bacterium of  claim 1 , wherein the bacterium expresses 4 of the proteins, and not TolR. 
     
     
         3 . A  Shigella  bacterium which does not express a TolR protein. 
     
     
         4 . A bacterium which is a ΔtolR strain of  Shigella.    
     
     
         5 . The bacterium of  claim 1 , wherein the bacterium does not express a native  Shigella  lipopolysaccharide. 
     
     
         6 . The bacterium of  claim 5 , wherein the bacterium does not express a native  Shigella  O antigen. 
     
     
         7 . The bacterium of  claim 4 , wherein the strain is a ΔtolRΔgalU strain. 
     
     
         8 . The bacterium of  claim 1 , wherein a stxA and/or stxB gene is inactivated. 
     
     
         9 . A  Shigella  bacterium which expresses TolA, TolB, TolQ, TolR and Pal proteins, wherein the TolA, TolQ, TolR and/or Pal protein (a) is located in the bacterium's inner or outer membrane, and (b) includes one or more amino acid sequence mutation(s) such that, compared to the same bacterium without said mutation(s), the bacterium releases greater quantities of outer membrane blebs when growing in culture medium. 
     
     
         10 . A  Shigella  bacterium in which one or more components of its Tol-Pal system has a modification such that, during growth in culture medium, the bacterium releases greater quantities of outer membrane blebs into the medium than the same bacterium lacking the modification, and which does not express: (i) a native  Shigella  lipopolysaccharide and/or (ii) a  Shigella  enteric toxin. 
     
     
         11 . A process for preparing  Shigella  blebs, comprising a step of separating the blebs from a culture medium comprising a bacterium of  claim 1 , which have been grown under conditions which permit the release of blebs into the medium by the bacteria. 
     
     
         12 . The process of  claim 11 , wherein the bacteria have been grown under iron-limiting conditions. 
     
     
         13 . A method of preparing a hyperblebbing  Shigella  bacterium, comprising a step of modifying gene(s) encoding one or more components of a starting bacterium's Tol-Pal system such that the modification causes the bacterium, when grown in culture medium, to release greater quantities of outer membrane blebs into the medium than the starting bacterium, and wherein the modification involves mutating one or more of the starting bacterium's TolA, TolB, TolQ, TolR and/or Pal genes. 
     
     
         14 . A bleb isolated or obtainable from a bacterium of  claim 1 . 
     
     
         15 . A composition comprising blebs that, during culture of a bacterium of  claim 1  are released into the culture medium. 
     
     
         16 . The composition of  claim 15 , which does not comprise any living and/or whole bacteria. 
     
     
         17 . A composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 μm filter of a culture medium in which a bacterium of  claim 1  has been grown. 
     
     
         18 . Culture media comprising bacteria of  claim 1 , which have been grown under conditions which permit the release of blebs into the medium by the bacteria. 
     
     
         19 . A  Shigella  strain which does not express a functional HtrB enzyme. 
     
     
         20 . The  Shigella  strain of  claim 19 , which is a htrB knockout. 
     
     
         21 . A  Shigella  bleb which includes one or more of: (a) a protein consisting of an amino acid sequence selected from SEQ ID NOs: 8 to 67; (b) a protein comprising an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 8 to 67 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 8 to 67, wherein the fragment comprises an epitope. 
     
     
         22 . A bleb-free immunogenic composition comprising a protein comprising: (a) amino acid sequences SEQ ID NOs 8 to 136; (b) an amino acid sequence having at least 85% identity to one of SEQ ID NOs: 8 to 136 and/or comprising a fragment of at least 7 consecutive amino acids of any one of SEQ ID NOs: 8 to 136 and comprising an epitope from one of SEQ ID NOs: 8 to 136. 
     
     
         23 . A process for culturing  Shigella , wherein iron-availability is limiting. 
     
     
         24 . A bleb isolated or obtainable from a bacterium obtainable by the method of  claim 13 . 
     
     
         25 . A bleb isolated or obtainable by the process of  claim 11 . 
     
     
         26 . A composition comprising blebs obtainable by the method of  claim 13  that are released into the culture medium. 
     
     
         27 . A composition comprising blebs, wherein the blebs are present in the filtrate obtainable after filtration through a 0.22 μm filter of a culture medium obtainable by the method of  claim 13  has been grown. 
     
     
         28 . Culture media comprising bacteria obtainable by the method of  claim 13 , which have been grown under conditions which permit the release of blebs into the medium by the bacteria.