Mutant fragments of OspA and methods and uses relating thereto
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. A nucleic acid encoding a polypeptide comprising a first disulfide bond-stabilized C-terminal fragment of an outer surface protein A (OspA),
wherein said 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 said polypeptide induces an immune response protective against a Borrelia infection, and wherein:
i) said 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) said 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
a) substitution of the wild-type amino acid at position 183+/−3 of SEQ ID NO: 8 by a cysteine, and
b) substitution of the wild-type amino acid at position 270+/−3 of SEQ ID NO: 8 by a cysteine,
wherein a disulfide bond between the introduced cysteines is present.
2. The nucleic acid of claim 1 , wherein the hybrid C-terminal OspA fragment consists of the amino acid sequence defined by SEQ ID NO: 1.
3. The nucleic acid of claim 1 , wherein said hybrid C-terminal OspA fragment consists of the amino acid sequence defined by SEQ ID NO: 51.
4. The nucleic acid of claim 1 , wherein said 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.
5. The nucleic acid of claim 1 , 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.
6. The nucleic acid of claim 5 , 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).
7. The nucleic acid of claim 1 , wherein the polypeptide comprises an E. coli -derived 1pp lipidation signal as defined by MKATKLVLGAVILGSTLLAG (SEQ ID NO: 15).
8. The nucleic acid of claim 1 , wherein 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.
9. The nucleic acid of claim 5 , wherein the polypeptide comprises a linker between the hybrid C-terminal OspA fragment and the second C-terminal OspA fragment, wherein said linker comprises the amino acid sequence GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 16).
10. The nucleic acid of claim 1 , further comprising a second hybrid C-terminal fragment of OspA.
11. The nucleic acid of claim 1 , wherein said polypeptide comprises the amino acid sequence of SEQ ID NO: 27.
12. The nucleic acid of claim 1 , wherein said polypeptide consists of the amino acid sequence of SEQ ID NO: 27.
13. A nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.
14. A vector comprising the nucleic acid of claim 1 .
15. A host cell comprising the vector of claim 14 .
16. The host cell of claim 15 , wherein said host cell is E. coli.
17. The host cell of claim 16 , wherein the E. coli is an E. coli BL21 cell.
18. A method for producing a polypeptide, comprising the following steps:
a) growing the host cell of claim 15 under conditions allowing for expression of said polypeptide, b) homogenizing said host cell to form a host cell homogenate, and c) subjecting the host cell homogenate to purification steps.
19. The method of claim 18 , wherein said purification steps comprise enriching the polypeptide in a lipid phase by phase separation and purifying over a gel filtration column.
20. The method of claim 18 , wherein said purification steps further comprise processing the polypeptide over a buffer exchange column.
21. The nucleic acid of claim 1 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 26.
22. The nucleic acid of claim 1 , wherein the nucleic acid consists of the nucleic acid sequence of SEQ ID NO: 26.
23. A nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 26.