Non-IgA fc binding forms of the Group B streptococcal beta antigens

Abstract

The invention relates to a mutant Cβ protein comprising the amino acid sequence A-X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 -B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X 1 -X 12 are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X 1 through X 12 , inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein. The invention also relates to a polynucleotide molecule encoding a mutant Cβ protein, as well as vectors comprising such polynucleotide molecules, and host cells transformed therewith. The invention also relates to a conjugate comprising the mutant Cβ protein covalently conjugated to a capsular polysaccharide. The invention also relates to a vaccine comprising at least one mutant Cβ protein of the invention and a pharmaceutically acceptable carrier. The invention also relates to a method of inducing an immune response in an animal, comprising administering the vaccine of the invention to an animal in a therapeutically effective amount.

What is claimed is:  
     
         1 . A polynucleotide molecule comprising a nucleotide sequence that encodes a mutant Cβ protein comprising the amino acid sequence A-X 1  X 2  X 3  X 4  X 5  X 6  X 7  X 8  X 9  X 10  X 11  X 12 -B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X 1 -X 12  are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X 1  through X 12 , inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein.  
     
     
         2 . The polynucleotide molecule of  claim 1 , wherein X 1 , X 5 , X 7 , X 8 , X 10 , X 11 , and X 12  are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), with the proviso that at least one of X 1 , X 5 , X 7 , X 8 , X 10 , X 11 , and X 12  is other than the wild type amino acid.  
     
     
         3 . The polynucleotide molecule of  claim 1 , wherein X 1  and X 11  are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.  
     
     
         4 . The polynucleotide molecule of  claim 1 , wherein X 1  and X 11  are Pro.  
     
     
         5 . The polynucleotide molecule of  claim 1 , wherein X 7  and X 12  are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.  
     
     
         6 . The polynucleotide molecule of  claim 1 , wherein X 7  and X 12  are Ala.  
     
     
         7 . The polynucleotide molecule of  claim 1 , wherein X 5 , X 7 , X 8 , X 10 , X 11  and X 12  are each a bond.  
     
     
         8 . A mutant Cβ protein comprising the amino acid sequence A-X 1  X 2  X 3  X 4  X 5  X 6  X 7  X 8  X 9  X 10  X 11  X 12 -B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X 1 -X 12  are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X 1  through X 12 , inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein.  
     
     
         9 . The protein of  claim 8 , wherein X 1 , X 5 , X 7 , X 8 , X 10 , X 11 , and X 12  are each selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), with the proviso that at least one of X 1 , X 5 , X 7 , X 8 , X 10 , X 11 , and X 12  is other than the wild type amino acid.  
     
     
         10 . The protein of  claim 8 , wherein X 1  and X 11  are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.  
     
     
         11 . The protein of  claim 8 , wherein X 1  and X 11  are Pro.  
     
     
         12 . The protein of  claim 8 , wherein X 7  and X 12  are selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, and a bond.  
     
     
         13 . The protein of  claim 8 , wherein X 7  and X 12  are Ala.  
     
     
         14 . The protein of  claim 8 , wherein X 5 , X 7 , X 8 , X 10 , X 11  and X 12  are each a bond.  
     
     
         15 . The mutant Cβ protein of  claim 8 , wherein the hydrophobic amino acid residues 1108-1116 are replaced by non-hydrophobic amino acids.  
     
     
         16 . The mutant Cβ protein of  claim 8 , wherein at least one of amino acid residues 521-541, inclusive, of Cβ is either (a) deleted or (b)) altered, so that the protein is not cleaved in this region when produced in  E. coli.    
     
     
         17 . The mutant Cβ protein of  claim 16 , wherein at least one of amino acid residues 533-541, inclusive, of Cβ is either (a) deleted or (b) altered.  
     
     
         18 . The mutant Cβ protein of  claim 16 , wherein at least one of amino acid residues 537-538 of Cβ is either (a) deleted or (b) altered.  
     
     
         19 . A polysaccharide-protein conjugate comprising the mutant Cβ protein of  claim 8  and a streptococcal capsular polysaccharide.  
     
     
         20 . A vector comprising the polynucleotide molecule of  claim 1 .  
     
     
         21 . A host cell transformed with the vector of  claim 20 .  
     
     
         22 . A vaccine comprising at least one mutant Cβ protein of  claim 8 , together with a pharmaceutically acceptable carrier.  
     
     
         23 . The vaccine of  claim 22 , wherein said mutant Cβ protein is conjugated to a polysaccharide.  
     
     
         24 . The vaccine of  claim 23 , wherein said polysaccharide to which said mutant Cβ protein is conjugated is selected from the group consisting of Group B streptococcal capsular polysaccharide types Ia, II, III and V.  
     
     
         25 . A combination vaccine comprising at least two Cβ protein-polysaccharide conjugates selected from the group consisting of Cβ-Ia, Cβ-II, Cβ-III and Cβ-V, together with a pharmaceutically acceptable carrier, wherein the Cβ portion of each conjugate is the mutant Cβ of  claim 8 .  
     
     
         26 . The vaccine of  claim 25 , said vaccine comprising Cβ-Ia, Cβ-II, Cβ-III and Cβ-V, together with a pharmaceutically acceptable carrier.  
     
     
         27 . A method of inducing an immune response in a mammal, comprising administering a vaccine comprising at least one mutant Cβ protein comprising the amino acid sequence A-X 1  X 2  X 3  X 4  X 5  X 6  X 7  X 8  X 9  X 10  X 11  X 12 -B, wherein A comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence starting from amino acid 177 and terminating at an amino acid between residue 1094 and 1127, inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and X 1 -X 12  are each selected independently from the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid found at the corresponding position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid positions are numbered from the first amino acid of the native amino acid sequence encoding said protein, with the proviso that at least one of X 1  through X 12 , inclusive, is other than the wild type amino acid, and wherein the LPXTG motif may be missing from the mutant Cβ protein, together with a pharmaceutically acceptable carrier, in an amount sufficient to induce an immune response in a mammal.  
     
     
         28 . The method of  claim 27 , wherein said mutant Cβ protein is conjugated to a streptococcal capsular polysaccharide.  
     
     
         29 . The method of  claim 28 , wherein said mammal is a human.