Mutant fragments of ospa and methods and uses relating thereto

Abstract

The present invention relates to compositions and methods for the prevention and treatment of Borrelia infection. Particularly, the present invention relates to a polypeptide comprising a hybrid C-terminal fragment of an outer surface protein A (OspA), a nucleic acid coding the same, an antibody specifically binding the same, a pharmaceutical composition (particularly for use as a medicament or in a method of treating or preventing a Borrelia infection) comprising the polypeptide and/or the nucleic acid and/or the antibody, a method of treating or preventing a Borrelia infection and a method of immunizing a subject.

1 .- 49 . (canceled) 
     
     
         50 . A method of producing a polypeptide, the method comprising:
 (a) introducing a vector encoding the polypeptide into a host cell;   (b) growing the host cell under conditions allowing for expression of the polypeptide;   wherein the polypeptide comprises a first disulfide bond-stabilized C-terminal fragment of an outer surface protein A (OspA),   wherein the first disulfide bond-stabilized C-terminal fragment is a hybrid C-terminal OspA fragment consisting of, from the N- to C-terminal direction, a fusion of a first and a second OspA portion from two different  Borrelia  strains, and wherein:   (i) the first OspA portion consists of amino acids 125-176 or amino acids 126-175 of OspA from a  Borrelia  strain that is not the corresponding fragment of  B. garinii , strain PBr OspA with SEQ ID NO: 8, wherein the numbering of amino acids is according to the numbering of corresponding amino acids of the full-length OspA of  B. burgdoferi  s.s., strain B31 with SEQ ID NO: 5; and   (ii) the second OspA portion consists of amino acids 176-274 or amino acids 177-274 of OspA from  B. garinii , strain PBr (SEQ ID NO: 8), wherein the second OspA portion differs from the corresponding wild-type sequence of SEQ ID NO: 8 at least by:
 (1) substitution of the wild-type amino acid at position 183+/−3 of SEQ ID NO: 8 by a cysteine, and 
 (2) substitution of the wild-type amino acid at position 270+/−3 of SEQ ID NO: 8 by a cysteine, 
 wherein a disulfide bond is present between the introduced cysteines. 
   
     
     
         51 . The method of  claim 50 , wherein the hybrid C-terminal OspA fragment comprises the amino acid sequence defined by SEQ ID NO: 1. 
     
     
         52 . The method of  claim 50 , wherein the hybrid C-terminal OspA fragment comprises the amino acid sequence defined by SEQ ID NO: 51. 
     
     
         53 . The method of  claim 50 , wherein the second OspA portion comprises a substitution of a threonine residue at position 233 of wild-type OspA of  B. garinii , strain PBr, as defined by SEQ ID NO: 8, with a proline residue. 
     
     
         54 . The method of  claim 50 , wherein the polypeptide further comprises a second disulfide bond-stabilized C-terminal OspA fragment; wherein said second disulfide bond-stabilized C-terminal OspA fragment consists of a C-terminal domain of an OspA from  B. burgdorferi  s.s.,  B. afzelii, B. bavariensis , or  B. garinii , which differs from the corresponding wild-type OspA sequence at least by the introduction of at least one disulfide bond, and wherein said second disulfide bond-stabilized C-terminal OspA fragment is not a hybrid C-terminal OspA fragment. 
     
     
         55 . The method of  claim 54 , wherein said at least one disulfide bond is formed by the substitution of the amino acid at position 182+/−3 of the wild-type sequence by a cysteine and by the substitution of the amino acid at position 269+/−3 of the wild-type sequence by a cysteine; and wherein the numbering of said amino acids is according to the numbering of corresponding amino acids of the full length OspA of  B. burgdorferi  s.s., strain B31 (SEQ ID NO: 5). 
     
     
         56 . The method of  claim 54 , wherein the polypeptide comprises a linker between the hybrid C-terminal OspA fragment and the second C-terminal OspA fragment, optionally wherein the linker comprises linker comprises the amino acid sequence 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 16) 
                 
                     
                   GTSDKNNGSGSKEKNKDGKYS 
                 
             
                
                
               
            
           
         
       
     
     
         57 . The method of  claim 50 , wherein the polypeptide comprises an  E. coli -derived 1pp lipidation signal as defined by MKATKLVLGAVILGSTLLAG (SEQ ID NO: 15); or
 the polypeptide comprises a lipidation site peptide led by an N-terminal cysteine residue as a site for lipidation, wherein said lipidation site peptide is defined by the amino acid sequence CSS.   
     
     
         58 . The method of  claim 50 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
         59 . The method of  claim 50 , wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 27. 
     
     
         60 . The method of  claim 50 , wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 26, optionally wherein the vector is pET28b(+). 
     
     
         61 . The method of  claim 50 , wherein the host cell is an  E. coli  cell, optionally an  E. coli  BL21 cell. 
     
     
         62 . The method of  claim 50 , further comprising
 (c) homogenizing the host cell to produce a host cell homogenate.   
     
     
         63 . The method of  claim 62 , further comprising
 (d) subjecting the host cell homogenate to one or more purification steps.   
     
     
         64 . The method of  claim 50 , wherein the one or more purification steps comprise enriching the polypeptide in a lipid phase separation and purifying the polypeptide over a gel filtration column. 
     
     
         65 . The method of  claim 64 , wherein the one or more purification steps further comprise processing the polypeptide over a buffer exchange column. 
     
     
         66 . A method for producing a pharmaceutical composition comprising combining a polypeptide with a pharmaceutically acceptable excipient,
 wherein the polypeptide comprises a first disulfide bond-stabilized C-terminal fragment of an outer surface protein A (OspA),   wherein the first disulfide bond-stabilized C-terminal fragment is a hybrid C-terminal OspA fragment consisting of, from the N- to C-terminal direction, a fusion of a first and a second OspA portion from two different  Borrelia  strains, wherein the polypeptide induces an immune response protective against a  Borrelia  infection, and wherein:   (i) the first OspA portion consists of amino acids 125-176 or amino acids 126-175 of OspA from a  Borrelia  strain that is not the corresponding fragment of  B. garinii , strain PBr OspA with SEQ ID NO: 8, wherein the numbering of amino acids is according to the numbering of corresponding amino acids of the full-length OspA of  B. burgdoferi  s.s., strain B31 with SEQ ID NO: 5; and   (ii) the second OSpA portion consists of amino acids 176-274 or amino acids 177-274 of OspA from  B. garinii , strain PBr (SEQ ID NO: 8), wherein the second OspA portion differs from the corresponding wild-type sequence of SEQ ID NO: 8 at least by:
 (1) substitution of the wild-type amino acid at position 183+/−3 of SEQ ID NO: 8 by a cysteine, and 
 (2) substitution of the wild-type amino acid at position 270+/−3 of SEQ ID NO: 8 by a cysteine, 
 wherein a disulfide bond is present between the introduced cysteines. 
   
     
     
         67 . The method of  claim 66 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 27. 
     
     
         68 . The method of  claim 66 , further comprising combining the polypeptide and pharmaceutically acceptable excipient with:
 (i) a second polypeptide comprising the amino acid sequence of SEQ ID NO: 29; and/or   (ii) a third polypeptide comprising the amino acid sequence of SEQ ID NO: 33.   
     
     
         69 . The method of  claim 66 , wherein the pharmaceutically acceptable excipient comprises L-methionine and/or aluminium hydroxide. 
     
     
         70 . The method of  claim 66 , wherein the pharmaceutical composition is a vaccine.