MUTANT FRAGMENTS OF OSPA AND METHODS AND USES RELATING THERETO
The present invention relates to a polypeptide comprising a mutant fragment of an outer surface protein A (OspA), a nucleic acid coding the same, a pharmaceutical composition (particularly for use as a medicament of in a method of treating or preventing a Borrelia infection) comprising the polypeptide and/or the nucleic acid, a method of treating or preventing a Borrelia infection and a method of immunizing a subject.
1 .- 48 . (canceled)
49 . A polypeptide comprising a mutant fragment of a Borrelia outer surface protein A (OspA), wherein said mutant OspA fragment comprises SEQ ID NO: 219, or a variant thereof, wherein said variant has at least 95% sequence identity to a wild-type serotype 2 OspA fragment defined by amino acid residues 126-273 of SEQ ID NO: 19, and wherein said variant differs from said wild-type serotype 2 OspA fragment at least by the addition of at least one disulfide bond.
50 . The polypeptide according to claim 49 , wherein said polypeptide comprises a heterodimer selected from the group consisting of Lip-S1 D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S1D4-S2D1 (SEQ ID NO: 197), and Lip-S2D1-S1D4 (SEQ ID NO: 204).
51 . The polypeptide according to claim 49 , wherein said polypeptide consists of a heterodimer selected from the group consisting of Lip-S1 D1-S2D1 (SEQ ID NO: 186), Lip-S2D1-S1D1 (SEQ ID NO: 192), Lip-S1D4-S2D1 (SEQ ID NO: 197), and Lip-S2D1-S1D4 (SEQ ID NO: 204).
52 . The polypeptide according to claim 49 , wherein said polypeptide is Lip-S1D1-S2D1 (SEQ ID NO: 186).
53 . A pharmaceutical composition comprising the polypeptide according to claim 49 .
54 . The pharmaceutical composition according to claim 53 , further comprising a pharmaceutically acceptable carrier or excipient.
55 . The pharmaceutical composition according to claim 54 , wherein said excipient is L-methionine and/or aluminium hydroxide.
56 . A method of treating or preventing against a Borrelia infection in a subject in need thereof comprising administering to the subject the polypeptide of claim 49 .
57 . The method according to claim 56 , wherein the Borrelia is selected from the group consisting of B. burgdorferi, B. garinii, B. afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi , and B. sinica.
58 . A method of treating or preventing against a Borrelia infection in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 53 .
59 . The method according to claim 58 , wherein the Borrelia is selected from the group consisting of B. burgdorferi, B. garinii, B. afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi , and B. sinica.
60 . A method for producing the polypeptide of claim 49 , comprising:
a) introducing a vector encoding the polypeptide into a host cell;
b) growing the host cell under conditions allowing for expression of said polypeptide;
c) homogenizing said host cell; and
d) subjecting the host cell homogenate to purification steps.
61 . The method according to claim 60 , wherein said vector is pET28b(+).
62 . The method according to claim 60 , wherein said host cell is Escherichia coli ( E. coli ).
63 . The method according to claim 62 , wherein said E. coli is an E. coli BL21 cell.
64 . The method according to claim 60 , wherein said purification steps comprise enriching the polypeptide in a lipid phase separation.
65 . The method according to claim 60 , wherein said purification steps comprise purifying the polypeptide over a gel filtration column.
66 . The method according to claim 60 , wherein said purification steps comprise processing the polypeptide over a buffer exchange column.