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.

Claims

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 TABLE-US-00008 (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.

Description

[0244] In connection with the present invention

[0245] FIG. 1 shows electrostatic potential isocontours of disulfide-bond-stabilized fragments of serotype 1-serotype 6 and B. valaisiana OspA (S1D1-S6D1 and BvaD1), as well as the mutant fusion OspA fragment of the invention consisting of amino acids 125-176 of B. valaisiana, strain VS116, and amino acids 177-274 of B. garinii, strain PBr, with an introduced disulfide bond (S3hybD1).

[0246] FIG. 2 shows the amino acid alignment of Lip-S4D1-S3hybD1 (SEQ ID NO: 27), the mutant serotype 3 OspA fusion fragment-containing heterodimer protein of the invention, with the heterodimer protein Lip-S4D1-S3D1 (SEQ ID NO: 31).

[0247] FIG. 3 schematically shows the production of mutant OspA fragment heterodimers according to the current invention.

[0248] FIG. 4 schematically represents the polypeptide components of a pharmaceutical composition of the current invention, an “improved combination vaccine”, comprising three different mutant OspA heterodimers, including Lip-S4D1-S3hybD1.

[0249] FIG. 5 shows the chemical structure of Pam.sub.3Cys, an example of a fatty acid substituted cysteine, such as would be found at the N-terminus of lipidated polypeptides of the current invention.

[0250] FIG. 6 shows IgG antibody titers to Borrelia OspA proteins of serotype 1-6 produced in mice in response to immunization with the improved heterodimer combination vaccine of the invention.

[0251] FIG. 7 shows the binding of antibodies from mice immunized with the improved heterodimer combination vaccine of the invention to the cell surface of Borrelia spirochetes of OspA serotypes 1-6.

[0252] Table 1 compares the purification yield of the Lip-S4D1-S3hybD1 heterodimer and the Lip-S4D1-S3D1 heterodimer.

[0253] Table 2 shows the protective capacity of the improved heterodimer combination vaccine of the invention against in vivo challenge with OspA serotypes 1, 2, 5 and 6 Borrelia.

[0254] The figures and tables which may be referred to in the specification are described below in more detail.

[0255] FIG. 1 Electrostatic potential simulation of the disulfide-bond-stabilized OspA fragments from serotypes 1-6 (S1D1-S6D1) and B. valaisiana (BvaD1) as well as the disulfide-bond-stabilized fusion OspA fragment (S3hybD1). Isocontours (+/−1 kT/e) are colored bright for negative charges and dark for positive charges, as a solid surface. The solvent-accessible surface is rendered as a wireframe. The white arrows indicate the position of the two extended clusters giving rise to electrostatic polarity on the same plane of the serotype 3 fragment. It can be seen that the surfaces of the hybrid OspA C-terminal fragment do not possess the extended electrostatic polar clusters as the S3D1 monomer.

[0256] FIG. 2 Amino acid sequence alignment of Lip-S4D1-S3hybD1 (SEQ ID NO: 27) and Lip-S4D1-S3D1 (SEQ ID NO: 31) heterodimer polypeptides, showing the consensus sequence. The Lip-S4D1-S3hybD1 heterodimer differs from the Lip-S4D1-S3D1 heterodimer by only 31 amino acids in total.

[0257] FIG. 3 Production of a mutant OspA heterodimer of the invention comprising two mutant OspA C-terminal fragments selected from different OspA serotypes of Borrelia sp. or a hybrid mutant OspA C-terminal fragment (A) Schematic representation of a nucleic acid encoding a lipidated mutant OspA heterodimer. The components, from 5′ to 3′, comprise the coding sequences for a lipidation signal sequence (Lip signal), a CSS peptide for N-terminal lipidation, a mutant C-terminal fragment of OspA with two non-native cysteines, a short linker peptide (LN1), followed by a second mutant OspA C-terminal fragment with two non-native cysteines. (B) The intermediate mutant OspA heterodimer polypeptide comprises the nascent product directly following translation of the nucleic acid construct. From the N- to the C-terminus, this polypeptide consists of a lipidation signal sequence (Lip signal), a CSS peptide for lipidation, a mutant OspA fragment with a non-native disulfide bond, a short linker peptide (LN1), followed by a second mutant OspA fragment with a non-native disulfide bond. (C) The final lipidated mutant OspA heterodimer polypeptide after post-translational modification. The heterodimer, from the N- to the C-terminus, consists of a CSS peptide with the N-terminal cysteine lipidated, a mutant OspA fragment stabilized by a disulfide bond, a linker peptide (LN1), and a second mutant OspA fragment stabilized by a disulfide bond. The lipidation signal sequence is cleaved off during post-translational modification of the polypeptide as shown.

[0258] FIG. 4 An example of a preferred pharmaceutical composition according to the current invention. Three mutant OspA heterodimers, each comprising two mutated OspA fragments selected from different Borrelia OspA serotypes or a hybrid mutant OspA C-terminal fragment (S3hybD1) are present in the composition, together providing OspA antigens from six different Borrelia OspA serotypes. Such a pharmaceutical composition enables simultaneous immunization against six Borrelia serotypes.

[0259] FIG. 5 Illustration of the chemical structure of Pam.sub.3Cys, an example of a fatty acid substitution of the N-terminal cysteine of full-length wild-type OspA protein as well as of lipidated mutant OspA fragment heterodimers of the invention. During post-translational modification of a full-length OspA protein or polypeptides of the invention, the N-terminal lipidation signal sequence is cleaved off and fatty acids, most commonly three palmitoyl moieties (“Pam.sub.3”), are enzymatically covalently attached to the N-terminal cysteine residue (the sulfur atom, “S”, is indicated by an arrow). The remaining residues of the polypeptide chain, which are located C-terminally from the Pam.sub.3Cys residue, are represented by “Xn”. (Modified from Bouchon, et al. (1997) Analytical Biochemistry 246: 52-61.)

[0260] FIG. 6 Antibody titers generated to all six serotypes of full-length OspA proteins. Mice were immunized three times with 3 μg each of the indicated combination vaccines: Lip-S1D1-S2D1, Lip-54D1-53D1 and Lip-S5D1-S6D1 together in a 1:1:1 ratio (“Het combo”); Lip-S1D1-S2D1, Lip-54D1-S3hybD1 and Lip-S5D1-S6D1 together in a 1:1:1 ratio (“Improved het combo”) or with Lip-Chimeric OspA ST1/ST2-His, Lip-Chimeric OspA ST5/ST3-His and Lip-Chimeric OspA ST6/ST4-His together in a 1:1:1 ratio (“Chimera combo”) at two week intervals and sera were collected at one week after the last dose. Titers of IgG antibodies to six different serotypes of full-length OspA proteins were determined by ELISA.

[0261] FIG. 7 Binding of antibodies from immunized mice to the cell surface of Borrelia spirochetes. Mice were immunized as above and sera were collected at one week after the last dose and pooled. Serial dilutions of the sera were tested for binding to the cell surface of Borrelia spirochetes via cell staining and flow cytometry. Fluorescence intensity values observed when staining with sera collected from control mice immunized with Al(OH).sub.3 adjuvant alone were subtracted to account for non-specific binding. (Borrelia used in the binding assay were: B. burgdorferi, OspA serotype 1, strain N40; B. afzelii, OspA serotype 2, strain PKo; B. garinii, OspA serotype 3, strain Fr; B. bavariensis, OspA serotype 4, strain Fin; B. garinii, OspA serotype 5, strain PHei; B. garinii, OspA serotype 6, strain KL11.)

EXAMPLES

Example 1. Molecular Modelling of the Hybrid Serotype 3 OspA C-Terminal Fragment

[0262] Motivation to Construct Hybrid OspA ST3 C-Terminal Fragments

[0263] The Lyme borreliosis combination vaccine as described in our previous application (WO2014/006226) is composed of three mutant OspA heterodimers. Short stabilized fragments from two different OspA serotypes (ST), derived from the C-terminal domain, are fused with a short linker to form a heterodimer. The three heterodimers are composed of OspA ST1-ST2, ST4-ST3 and ST5-ST6. For improvement of the immunogenicity of the heterodimers, a signal sequence for lipidation is added in analogy with mature full-length OspA which is a lipoprotein.

[0264] The lipidated heterodimer composed of OspA ST4-ST3 (Lip-S4D1-S3D1; SEQ ID NO: 31) proved to be less soluble than the two other heterodimers, which results in low recovery during purification. This problem can mostly be attributed to the short stabilized OspA ST3 portion since this protein, as a lipidated monomer, cannot be expressed and purified.

[0265] Molecular Modelling

[0266] A comparative structural investigation of the stabilized monomers was untertaken in silico to elucidate the compatibility of the folds between the monomer models compared to the OspA crystal structure (PDB:1OSP; Li H, Dunn J J, Luft B J, Lawson C L (1997) Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab. Proc Natl Acad Sci 94: 3584-3589) and to compare their surface properties with the ST3 type monomer. The crystal structure represents serotype 1 of Borrelia burgdorferi B31, and shows OspA bound with its N-terminus, to the murine antibody Fab 184.1. Homology structure models of the six short stabilized OspA monomers were constructed starting with the ST1 OspA crystal structure and available homology models (SwissModel; Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Res 37: D387-392), which were then modified to incorporate the stabilizing disulfide bonds and sequence changes where applicable (The PyMOL Molecular Graphics System, Version Open-Source, Schrödinger LLC).

[0267] The electrostatic potential isocontours of all six short stabilized OspA monomers were simulated with the adaptive Poisson-Boltzmann solver (APBS; Baker N A, Sept D, Joseph S, Holst M J, McCammon J A (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci 98: 10037-10041, pdb2pqr; Dolinsky T J, Czodrowski P, Li H, Nielsen J E, Jensen J H, et al. (2007) PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res 35: W522-525) to determine if the short stabilized OspA ST3 protein has a pattern in surface electrostatics which deviates from the other OspA STs, which could explain the solubility problem of short stabilized OspA ST3 (“S3D1”, FIG. 1). A significant polarity in the charge distribution was observed on one side of S3D1 (see arrows), which was not observed in any of the other short stabilized OspA STs.

[0268] An electrostatic potential simulation was also performed with short stabilized OspA from B. valaisiana (“BvaD1”, FIG. 1). The BvaD1 OspA fragment displayed a variant polarity of the electrostatic potential isocontours on the surface, which made it a potential candidate building block for a partial segmental exchange with the aim to modify the overall surface to not show any significant extended clusters of extended electrostatic polarity as found in S3D1. The preferred link between the serotype 3 part and the exchanged part from B. valaisiana in the hybrid monomer was chosen to replace the N-terminal beta-sheet of the monomer and to leave two beta sheets and the C-terminal helix of the serotype 3 portion intact, under retention of the overall fold. The latter condition depends largely on the steric compatibility of the densely packed hydrophobic residues in the core of the molecule. Fold compatibility for the model of the hybrid stabilized monomer, S3hybD1, was verified with molecular mechanics simulation (Gromacs; Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, et al. (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29: 845-854).

[0269] Application in the Form of Heterodimers Containing the Hybrid OspA Fragment

[0270] As a result of the electrostatic potential simulations, a new heterodimer containing the experimental fusion of fragments OspA proteins from B. valaisiana and B. garinii (S3hybD1, FIG. 1), was cloned: Lip-S4D1-S3hybD1. In addition to the changes of the first one-third of the serotype 3 OspA fragment in the heterodimer, 54D1-S3hybD1, compared with Lip S4D1-S3D1, an amino acid substitution at position 233 (P233T, amino acid nomenclature according the immature full-length OspA; SEQ ID NO: 8) of OspA ST3 was introduced.

[0271] As illustrated in further detail below, this new heterodimer shows substantially improved solubility as well as immunogenicity against Borrelia expressing serotype 3 OspA when purified and used to immunize mice.

Example 2. Purification and Formulation of Lipidated Mutant OspA Fragment Heterodimers

[0272] Cloning and Expression of Lipidated Non-His-Tagged Mutant OspA Fragment Heterodimers

[0273] The fusion OspA monomer B. valaisiana/B. garinii strain VS116/PBr was codon-optimized for E. coli expression by GeneArt (Germany) The lipidation signal sequence added to the N-terminal end was derived from the E. coli major outer membrane lipoprotein, Lpp, and was followed directly C-terminally by a CSS peptide to provide an N-terminal cysteine for lipidation. The improved heterodimer construct was generated by fusing the mutant serotype 4 OspA fragment and the hybrid serotype 3 OspA fragment via the linker sequence “LN1”. Gene fragments were cloned into the pET28b(+) vector (Novagen, USA), and the stabilized heterodimers were expressed in BL21(DE3) cells (Invitrogen, USA). Cells were collected after 4 h by centrifugation and the pellet was stored at −70° C. for up to 12 months prior to further processing.

[0274] Purification of Lipidated Mutant OspA Fragment Heterodimers

[0275] Cells were disrupted mechanically by high-pressure homogenization and the lipidated mutant OspA fragment heterodimers, Lip-S4D1-S3D1 and Lip-S4D1-S3hybD1, were enriched in the lipid phase by phase separation, using Triton X-114 as detergent. Subsequently, the diluted detergent phase was subjected to anion exchange chromatography (Q-sepharose; GE Healthcare, United Kingdom) operated in non-binding mode. The resulting flow-through was loaded on a hydroxyapatite column (Bio-Rad, USA) and the lipidated proteins were eluted from the column by a linear salt gradient. The eluate was subjected to further purification over a DEAE-Sepharose column (GE Healthcare) in non-binding mode followed by gel filtration column (Superdex 200, GE Healthcare) for buffer exchange. The lipidated mutant OspA heterodimer peaks were pooled on the basis of the analytical size exclusion column and SDS-PAGE. After sterile filtration, the purified heterodimers were stored at −20° C. until formulation.

[0276] Formulation of the Combination Vaccine

[0277] Studies regarding the formulation of the combination vaccine of the invention were carried out in order to optimize stability. Different types of buffers and stabilizers were tested at various concentrations in combination with aluminium hydroxide and antigen, as described in our previous application WO2014/006226. An optimal formulation of 40 μg/mL each of three heterodimers (120 μg total protein), 10 mM sodium phosphate, 150 mM sodium chloride, 10 mM L-Methionine, 5% Sucrose, 0.05% Tween 20 (polysorbate 20) and 0.15% (w/v) aluminium hydroxide at pH 6.7±0.2 was determined.

[0278] Results

[0279] The improved heterodimer, Lip-S4D1-S3hybD1 showed an about 4-fold higher yield in terms of mg/g biomass, a significant improvement over Lip-S4D1-S3D1. Additionally, comparable purity of the improved heterodimer preparation was achievable with one less chromatography step. (See Table 1.)

TABLE-US-00005 TABLE 1 Improved yield of heterodimer Lip S4D1-S3hybD1 compared with Lip-S4D1-S3D1. Number of Yield (mg/g Purity (%) Purity (%) Purity (%) Chromatography Construct biomass) RP-HPLC SEC-HPLC SDS-PAGE steps Lip-S4D1-S3D1 0.35 88 95 90 5 Lip-S4D1-S3hybD1 2.5 82 96 97 4

Example 3. Immunogenicity of Lipidated Mutant OspA Fragment Heterodimers of Different Serotypes

[0280] Immunization of mice Female C.sub.3H/HeN mice were used for all studies. Prior to immunizations, groups of ten mice were bled via the facial vein and pre-immune sera were prepared and pooled. Three s c immunizations of 100 μL each were administered at two week intervals. Each dose contained 1 μg of the respective heterodimer proteins. For the improved heterodimer combination vaccine: Lip-S1D1-S2D1 (SEQ ID NO: 29), Lip-S4D1-S3hybD1 (SEQ ID NO: 27) and Lip-S5D1-S6D1 (SEQ ID NO: 33); for the heterodimer combination vaccine of the previous invention: Lip-S1D1-S2D1 (SEQ ID NO: 29), Lip-54D1-53D1 (SEQ ID NO: 31) and Lip-S5D1-S6D1 (SEQ ID NO: 33) and for the chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His (Seq ID No: 40), Lip-Chimeric OspA ST5/ST3-His (Seq ID No: 41) and Lip-Chimeric OspA ST6/ST4-His (SEQ ID NO: 42). All combination vaccines were formulated with aluminium hydroxide (Al(OH).sub.3) at a final concentration of 0.15%. One week after the third immunization, blood was collected from the facial vein and immune sera prepared. In each experiment, one group immunized with PBS formulated with Al(OH).sub.3 was included as a negative control (placebo group). All animal experiments were conducted in accordance with Austrian law (BGB1 Nr. 501/1989) and approved by “Magistratsabteilung 58”.

[0281] OspA ELISA

[0282] ELISA plates (Maxisorp, Nunc, Denmark) were coated with 50 ng (1 μg/mL) protein diluted in coating buffer (PBS) per well and incubated at 4° C. for 16 to 72 hours. The coating antigens were C-terminally His-tagged full length lipidated OspA ST1-6. The coating buffer was discarded and 100 μL blocking buffer (1% BSA, 0.5% Tween-20, PBS) was added and incubated at ambient temperature for 1-2 hours. Plates were washed three times with 300 μL (overflow) PBST (0.1% Tween-20, PBS). Five-fold dilutions of the sera were made in blocking buffer and 50 μL were added to each well and incubated for 1 hour at ambient temperature. Plates were washed three times with 300 μL (overflow) PBST. The secondary antibody (horseradish peroxidase [HRP]-conjugated rabbit anti-mouse IgG, DAKO, Denmark) was diluted 1:2000 in blocking buffer and 50 μL was added to each well and incubated for 1 hour at ambient temperature. Plates were washed three times with 300 μL (overflow) PBST. ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Sigma-Aldrich, USA) was used as substrate for HRP, 50 μL of ABTS was added to each well and incubated for 15 minutes in the dark at ambient temperature. The reaction was stopped by the addition of 50 μL 1% SDS and the absorbance was read at 405 nm. A plate was regarded as valid when the absorbance of the blank was below 0.1. A sample was valid when the lowest dilution had an absorbance above 1.0 and the highest dilution was below 0.1. When these criteria were met, the half-max titer was determined. The half-max titer is the reciprocal of the dilution that corresponds to the mean absorbance between the highest and lowest dilutions.

[0283] Flow Cytometry

[0284] Spirochetes (1×10.sup.6) were mixed with an equal volume of 4% paraformaldehyde and incubated for 2 hours at room temperature in a 96-well plate (Nunclon 96U, Nunc). The plate was centrifuged for 5 minutes at 2,000 g and the supernatant was discarded. Cells were washed with 150 μL HBSS with 2% BSA (HBSS-B), centrifuged as above, and the supernatant was discarded. Mouse sera were heat inactivated by incubating them at 56° C. for 35 minutes. Heat inactivated sera were diluted in HBSS-B and sterile filtered by centrifuging at 4,000 g for 3 minutes using Costar spin-X centrifuge tube filters (0.22 μm, Corning, USA). Spirochetes were dissolved in 100 μL serum and incubated for 45 minutes at room temperature. The plate was centrifuged for 15 minutes at 2,000 g and the supernatant was discarded. The cells were washed once with 150 μL HBSS-B and then resuspended in 100 μL HBSS-B. One microliter secondary antibody (PE conjugated goat anti-mouse IgG, Beckman Coulter, USA) was added to the cells and incubated at room temperature for 45 minutes in the dark. Spirochetes were washed once with 150 μL HBSS-B and then resuspended in 200 μL HBSS containing 2.5 μM SYTO-17 DNA dye and incubated for 10 minutes at room temperature in the dark. The stained spirochetes were pelleted by centrifuging for 5 minutes at 2,000 g and subsequently resuspended in 200 μL HBSS. Labelled spirochetes were measured with an FC500 (Beckman Coulter) flow cytometer, gated for SYTO-17 positive events.

[0285] Results

[0286] Two different OspA heterodimer formulations (“het combo” and “improved het combo”) as well as an OspA chimera combination (“chimera combo”) were tested for immunogenicity in mice. Hyperimmune sera were analysed by ELISA for reactivity against full-length OspA (coating antigen) as well as for surface binding to Borrelia strains expressing different OspA serotypes (ST1 to ST6).

[0287] The ELISA results indicated that all vaccine combinations stimulated antibody responses to all six OspA serotypes (see FIG. 6). It is especially noteworthy with regard to the current invention that the improved OspA heterodimer combination vaccine resulted in higher levels of antibodies specific to serotype 3 OspA in comparison with the OspA heterodimer combination vaccine of the previous invention, whereas antibody levels to other OspA serotypes were comparably stimulated by both vaccines.

[0288] Binding of antibodies from hyperimmune mouse sera directly to borrelia spirochetes was observed in the case of Borreliae expressing all six OspA serotypes (see FIG. 7), indicating that the antibodies generated in response to all of the antigens are functionally active and can bind native OspA in situ. The fluorescence intensity was linear over a large range of serum dilutions. The fluorescence intensity observed in response to the improved heterodimer combination vaccine to spirochetes was comparable to those observed in response to the heterodimer combination vaccine and the chimera combination vaccine. Notably, with regard to binding to serotype 3 OspA borrelia, the antibodies generated by immunization with the improved vaccine were superior to antibodies generation in response to both of the other combination vaccines.

Example 4. Protective Capacity of the Improved Heterodimer Combination Vaccine Against In Vivo Borrelia Challenge

[0289] Immunization of Mice

[0290] Female C.sub.3H/HeN (H-2.sup.k) mice were used for all studies (Janvier, France). Prior to each challenge, groups of five 8-week-old mice were bled via the tail vein and pre-immune sera were prepared and pooled. Three subcutaneous (s c) immunizations of 100 μL were administered at two week intervals at the doses indicated in Table 2. Both the improved heterodimer combination vaccine and the chimera combination vaccine included three proteins at a ratio of 1:1:1 as described in Example 3. All formulations included aluminium hydroxide (Al(OH).sub.3) at a final concentration of 0.15%. One week after the third immunization, blood was collected and hyper-immune sera were prepared. In each experiment, one group injected with Al(OH).sub.3 alone (in formulation buffer or PBS) was included as a negative control and one group of mice immunized with the wild-type full-length lipidated OspA protein from the appropriate OspA serotype served as a positive control group (B. burgdorferi strain B31 (OspA serotype 1, SEQ ID NO: 34), B. afzelii strain K78 (OspA serotype 2, SEQ ID NO: 35), B. garinii strain PHei (OspA serotype 5, SEQ ID NO: 38) or B. garinii strain DK29 (OspA serotype 6, SEQ ID NO: 39)). All animal experiments were conducted in accordance with Austrian law (BGB1 Nr. 501/1989) and approved by “Magistratsabteilung 58”.

[0291] Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

[0292] Two weeks after the last immunization, mice were challenged s.c. with spirochetes diluted in 100 μL growth medium (BSKII). B. burgdorferi strain ZS7 expressing OspA serotype 1 (experiments 1 and 2), B. garinii strain PHei expressing OspA serotype 5 (experiments 10 to 13) or B. garinii strain Ma expressing OspA serotype 6 (experiments 14 to 17) were used for challenge. The challenge doses were strain-dependent and dependent on the virulence of the individual strains, which was assessed by challenge experiments for determination of ID.sub.50. Doses employed for needle challenge experiments ranged from 20 to 50 times the ID.sub.50. Prior to each challenge, OspA expression was verified by flow cytometry (see example 3). Challenge of mice was only performed with cultures where >80% of cells were positive for OspA expression.

[0293] Challenge of Immunized Mice with Ticks Infected with B. burgdorferi or B. afzelii (“Tick Challenge”)

[0294] Two weeks after the last immunization, mice were challenged with ticks harboring B. burgdorferi strain Pra4 expressing OspA serotype 1 (experiment 3), B. burgdorferi strain Praa1 expressing OspA serotype 1 (experiments 4 and 5) or B. afzelii expressing OspA serotype 2 (experiments 6 to 9). In order to facilitate tick infection of the immunized mice, the hair of the back of each mouse was removed with Veet® Cream (Reckitt Benckiser, United Kingdom) and a small ventilated container was glued to the skin with super glue (Pattex, Germany) Thereafter, two to three I. ricinus nymphs infected with B. burgdorferi strain Pra1 or Pra 4 or B. afzelii strain IS1 were applied per mouse and allowed to attach and feed until they were fully engorged and dropped off. The feeding status was monitored daily for each individual tick. Only those mice from which at least one fully- or almost fully-fed tick was collected were included in the final readout.

[0295] Sacrifice of Mice and Collection of Material

[0296] Four or six weeks after needle or tick challenge, respectively, mice were sacrificed by cervical dislocation. Blood was collected by orbital bleeding and final sera were prepared and used for VlsE ELISA and/or western blot to determine infection status. In addition, the urinary bladder from each mouse was collected and DNA was extracted and subjected to quantitative PCR (qPCR) for identification of Borrelia.

[0297] ELISA with the Invariable Region 6 (IR6) of VlsE

[0298] A biotinylated 25-mer peptide (MKKDDQIAAAMVLRGMAKDGQFALK, SEQ ID NO: 59) derived from the sequence of B. garinii strain IP90 was used for the analysis (Liang F T, Alvarez A L, Gu Y, Nowling J M, Ramamoorthy R, Philipp M T. An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi. J Immunol. 1999; 163:5566-73). Streptavidin pre-coated 96-well ELISA plates (Nunc), were coated with 100 μL/well (1 μg/mL) peptide in PBS supplemented with 0.1% Tween (PBS/0.1T). The plates were incubated overnight at 4° C. After coating with the peptide, the plates were washed once with PBS/0.1T. The plates were then blocked for one hour at room temperature (RT) with 100 μL/well of PBS+2% BSA, before being washed again with PBS/0.1T. Reactivity of post-challenge sera (final sera) to the peptide was tested at 1:200 and 1:400 dilutions in PBS+1% BSA. Plates were incubated for 90 minutes at RT before being washed three times with PBS/0.1T. Each well then received 50 μL of 1.3 μg/mL polyclonal rabbit anti-mouse IgG conjugated to HRP (Dako) in PBS+1% BSA. The plates were then incubated for 1 hour at RT. After three washes with PBS/0.1T, ABTS (50 μL/well) was added as substrate (Sigma-Aldrich) and color was allowed to develop for 30 minutes. Absorbance was measured at 405 nm. All sera were tested in duplicate. Negative controls included PBS instead of sera as well as plates not coated with the peptide. Sera from mice shown to be culture positive for borrelia infection were used as positive controls.

[0299] DNA Extraction and Purification

[0300] The urinary bladder from each mouse was subjected to DNA extraction and purification using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions with the following modification. Each urinary bladder was digested overnight at 60° C. using 100 recombinant Proteinase K (PCR grade; 14-22 mg/mL, Roche). The DNA was eluted in 50 μL sterile deionized water and stored at −20° C. As a negative control, every tenth sample was followed by one empty purification column in each DNA extraction and purification.

[0301] qPCR Targeting recA

[0302] Oligonucleotide primers were designed for the recA gene in a manner that they could be used in qPCR for identification of all relevant Borrelia species causing Lyme borreliosis (forward: CATGCTCTTGATCCTGTTTA, SEQ ID NO: 57, reverse: CCCATTTCTCCATCTATCTC, SEQ ID NO: 58). The recA fragment was cloned from the B. burgdorferi s.s. strain N40 into pET28b(+), to be used as standard in each reaction. The chromosomal DNA extracted from mouse urinary bladders was diluted 1:4 in water in order to reduce matrix effects observed with undiluted DNA. A master mix consisting of 10 μL SSoAdvanced™ SYBR® Green Supermix, 0.3 μL of each primer (10 μM), and 7.4 μL water was prepared for each experiment. Eighteen μL of master mix was mixed with 2 μL of the diluted DNA extracted from urinary bladder in micro-titer plates and the DNA was amplified using a CFX96 real-time PCR detection system (Bio-Rad). The DNA was denatured for 3 minutes at 95° C., followed by 50 cycles of 15 seconds at 95° C. and 30 seconds at 55° C. After amplification, the DNA was prepared for melting curve analysis by denaturation for 30 seconds at 95° C. followed by 2 minutes at 55° C. The melting curve analysis was performed by 5 seconds incubation at 55° C., with a 0.5° C. increase per cycle, and 5 seconds at 95° C. On each plate, four no-template controls (NTC) were included as well as a standard curve in duplicate with template copy numbers ranging from 10 to 10,000.

[0303] Western Blot

[0304] Binding of final sera to whole cell lysates from borrelia belonging to the corresponding OspA serotype was analyzed by western blot. Briefly, 2.5 μg of spirochete lysate per mouse sera to be analyzed was separated by SDS-PAGE under reducing conditions using 4-12% Tris-Glycine ZOOM gels (Invitrogen). Separated proteins were transferred onto a nitrocellulose membrane using the iBlot® Dry blotting system (Invitrogen). After blocking in 5% milk for 1 hour, final sera were added at a 1:2000 dilution and incubated at +4° C. overnight. The membranes were then washed three times with PBS/0.1T followed by a one hour incubation in polyclonal rabbit anti mouse IgG conjugated to HRP (Dako) diluted 1:10,000. The immunoblots were visualized with Amersham ECL Plus™ Western blotting detection reagents (GE Healthcare) and Kodak BioMax films (Kodak).

[0305] Infection Readout

[0306] The final infection readout was based on two separate methods: detecting the presence of Borrelia-specific antibodies (western blot and VlsE ELISA) and presence of Borrelia DNA (qPCR targeting recA). In experiments where B. burgdorferi strain ZS7 was used for challenge (experiments 1 and 2), western blot together with qPCR were applied. In all other experiments, VlsE ELISA and qPCR were used. There was a high consistency between the two methods (>95%); therefore a mouse was regarded as infected when at least one of the two methods was positive. Statistical significance was determined by Fisher's exact test (two-tailed).

[0307] Results

[0308] The improved heterodimer combination vaccine was tested for protective capacity against Borrelia challenge. The results of these experiments are summarized in Table 2 Immunized mice were challenged with B. burgdorferi s.s. (OspA serotype 1, strain ZS7, needle challenge, Experiments 1 and 2 or strains Pra1 or Pra 4, tick challenge, Experiments 3 or 4 and 5, respectively), B. afzelii (OspA serotype 2, strain IS1, tick challenge, Experiments 6-9), B. garinii (OspA serotype 5, strain PHei, needle challenge, Experiments 10-13) or B. garinii (OspA serotype 6, strain Ma, needle challenge, Experiments 14-17). In some experiments, other OspA-based antigens, such as the chimera combination vaccine or a lipidated full-length OspA protein, were included. A group of mice immunized with PBS or formulation buffer combined with Al(OH).sub.3 served as a placebo (adjuvant alone) control group in each experiment.

[0309] The protection data from the 17 experiments are summarized in Table 2. In all experiments, a high level of infection was seen in all placebo groups. Additionally, a low infection rate was observed in the groups receiving the corresponding full length OspA protein, with the exception of the full-length OspA serotype 6, wherein only partial protection was observed (experiments 14 to 17). These results validate the experimental set-up and readout methods.

[0310] The improved heterodimer combination vaccine conferred significant protection (p-values<0.05), at a 3 μg dose, when mice were challenged with in vitro grown B. burgdorferi s.s. or B. garinii (OspA serotype 5 or 6), or ticks harboring B. burgdorferi or B. afzelii. Furthermore, when different immunization doses were assessed for vaccine efficacy, highly significant protection (p-values<0.01) could be shown when 0.03 μg of the improved heterodimer combination vaccine was administered and the mice were challenged with B. afzelii or B. garinii (OspA serotype 5 or 6) (Experiment 8, 9, 12, 13 and 16). In summary, the improved heterodimer combination vaccine induced protective immunity against three Borrelia species (B. burgdorferi, B. afzelii and B. garinii) including four clinically relevant OspA serotypes (1, 2, 5 and 6), as shown in mouse models using either in vitro grown spirochetes or infected ticks for challenge.

[0311] Protection against serotypes 5 and 6 comparable to that conferred by the improved heterodimer combination vaccine was also observed in mice immunized with the chimera combination vaccine (data not shown).

TABLE-US-00006 TABLE 2 Protective capacity of the improved mutant OspA heterodimer combination vaccine of the invention against OspA serotype 1, serotype 2, serotype 5 and serotype 6 Borrelia challenge. Groups of mice were immunized three times with the indicated doses of immunogen or Al(OH).sub.3 adjuvant alone at two-week intervals. Immunogens used were a 1:1:1 combination of the mutant OspA heterodimers Lip-S1D1-S2D1, Lip-S4D1-S3hybD1 and Lip-S5D1-S6D1 (“Improved heterodimer combination vaccine”), a 1:1:1 combination of Lip-Chimeric OspA ST1/ST2-His, Lip- Chimeric OspA ST5/ST3-His and Lip-Chimeric OspA ST6/ST4-His (“Chimera combination vaccine”) and Lip-OspA1-His (lipidated full-length OspA protein from B. burgdorferi strain B31) or Lip-OspA2-His (lipidated full-length OspA protein from B. afzelii strain K78), Lip-OspA5-His (lipidated full-length OspA protein from B. garinii strain PHei) or Lip-OspA6-His (lipidated full- length OspA protein from B. garinii strain DK29). Immunized mice were challenged s.c. two weeks after the last immunization with the indicated borrelia species using a syringe (B. burgdorferi strain ZS7, B. garinii strain PHei or B. garinii strain Ma) or using ticks (B. burgdorferi strain Pra1 or Pra4 or B. afzelii strain IS1). A Protection against needle challenge with serotype 1 OspA borrelia by chimera combination vaccine and improved heterodimer combination vaccine (one dose: 3 μg) Experiment 1: Experiment 2: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA1-His (SEQ ID NO: 34) 3 × 1.0 μg B. burgdorferi  0/10 (<0.0001)  1/10 (<0.0001) (OspA serotype 1) strain ZS7 Chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His 3 × 1.0 μg B. burgdorferi  0/10 (<0.0001)  0/10 (<0.0001) (Seq ID No: 40) (OspA serotype 1) Lip-Chimeric OspA ST5/ST3-His 3 × 1.0 μg strain ZS7 (Seq ID No: 41) Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg (Seq ID No: 42) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. burgdorferi  0/10 (<0.0001)  0/10 (<0.0001) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg (OspA serotype 1) Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg strain ZS7 Al(OH).sub.. adjuvant alone — B. burgdorferi  9/9 10/10 (OspA serotype 1) strain ZS7 B Protection against tick challenge with serotype 1 OspA borrelia by chimera combination vaccine and improved heterodimer combination vaccine (one dose: 3 μg) Experiment 3: Experiment 4: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA1-His (SEQ ID NO: 34) 3 × 1.0 μg Tick challenge with  0/4 (0.0475)  1/9 (0.0216) B. burgdorferi (OspA serotype 1) strain Pra4 (Exp. 3) or Pra1 (Exp. 4) Chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His 3 × 1.0 μg Tick challenge with  0/8 (0.0060)  0/7 (0.0093) (Seq ID No: 40) B. burgdorferi Lip-Chimeric OspA ST5/ST3-His 3 × 1.0 μg (OspA serotype 1) (Seq ID No: 41) strain Pra4 (Exp. 3) Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg or Pra1 (Exp. 4) (Seq ID No: 42) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg Tick challenge with  0/5 (0.0260)  0/7 (0.0093) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg B. burgdorferi Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg (OspA serotype 1) strain Pra4 (Exp. 3) or Pra1 (Exp. 4) Al(OH).sub.3 adjuvant alone — Tick challenge with  5/6  5/6 B. burgdorferi (OspA serotype 1) strain Pra4 (Exp. 3) or Pra1 (Exp. 4) C Protection against tick challenge with serotype 1 OspA borrelia by improved heterodimer combination vaccine (decreasing doses: 3 μg and 0.3 μg) Experiment 5: Infected/Total Immunogen Dose Challenge (p-value) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg Tick challenge with 1/7 (0.0174) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg B. burgdorferi Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg (OspA serotype 1) strain Pra16 Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.1 μg Tick challenge with 1/9 (0.0059) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.1 μg B. burgdorferi Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg (OspA serotype 1) strain Pral Al(OH).sub.3 adjuvant alone — Tick challenge with 7/8 B. burgdorferi (OspA serotype 1) strain Pral D Protection against tick challenge with serotype 2 OspA borrelia by chimera combination vaccine and improved heterodimer combination vaccine (one dose: 3 μg) Experiment 6: Experiment 7: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA2-His (SEQ ID NO: 35) 3 × 1.0 μg Tick challenge with  0/8 (0.0016)  0/9 (0.0002) B. afzelii (OspA serotype 2) strain IS1 Chimera combination vaccine: Lip-Chimeric OspA ST1/ST2-His 3 × 1.0 μg Tick challenge with  0/7 (0.0025)  0/9 (0.0002) (Seq ID No: 40) B. afzelii (OspA Lip-Chimeric OspA ST5/ST3-His 3 × 1.0 μg serotype 2) strain (Seq ID No: 41) IS1 Lip-Chimeric OspA ST6/ST4-His 3 × 1.0 μg (Seq ID No: 42) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg Tick challenge with  0/9 (0.0010)  0/7 (0.0003) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg B. afzelii (OspA Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg serotype 2) strain IS1 Al(OH).sub.3 adjuvant alone — Tick challenge with  5/5  8/8 B. afzelii (OspA serotype 2) strain IS1 E Protection against tick challenge with serotype 2 OspA borrelia by improved heterodimer combination vaccine (decreasing doses: 0.03 μg and 0.003 μg) Experiment 8: Experiment 9: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.01 μg Tick challenge with  0/9 (0.0004)  2/7 (0.0096) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.01 μg B. afzelii (OspA Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.01 μg serotype 2) strain IS1 Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.001 μg Tick challenge with  7/10 (n.s.)  1/8 (0.0008) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.001 μg B. afzelii (OspA Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.001 μg serotype 2) strain IS1 Al(OH).sub.3 adjuvant alone — Tick challenge with  6/6  9/9 B. afzelii (OspA serotype 2) strain IS1 F Protection against needle challenge with serotype 5 OspA borrelia by improved heterodimer combination vaccine (one dose: 3 μg) Experiment 10: Experiment 11: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA5-His (SEQ ID NO: 38) 3 × 1.0 μg B. garinii (OspA  0/10 (<0.0001)  0/10 (<0.0001) serotype 5) strain PHei) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA  0/10 (<0.0001)  1/10 (<0.0001) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg PHei) Al(OH).sub.3 adjuvant alone — B. garinii (OspA 10/10 10/10 serotype 5) strain PHei) G Protection against needle challenge with serotype 5 OspA borrelia by improved heterodimer combination vaccine (decreasing doses: 3 μg, 0.3 μg and 0.03 μg) Experiment 12: Experiment 13: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA5-His (SEQ ID NO: 38) 3 × 1.0 μg B. garinii (OspA  0/10 (0.0007)  0/10 (<0.001) serotype 5) strain PHei) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA  0/10 (<0.0007)  1/10 (0.0002) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg PHei) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.1 μg B. garinii (OspA  0/10 (<0.0007)  0/10 (<0.0001) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.1 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg PHei) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.01 μg B. garinii (OspA  2/10 (<0.0219)  3/10 (0.0047) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.01 μg serotype 5) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.01 μg PHei) Al(OH).sub.3 adjuvant alone — B. garinii (OspA  8/10  9/9 serotype 5) strain PHei) H Protection against needle challenge with serotype 6 OspA borrelia by improved heterodimer combination vaccine (one dose: 3 μg) Experiment 14: Experiment 15: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA6-His (SEQ ID NO: 39) 3 × 1.0 μg B. garinii (OspA  4/10 (0.0108)  6/10 (n.s.) serotype 6) strain Ma) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA  0/10 (<0.0001)  0/10 (0.0007) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg Ma) Al(OH).sub.3 adjuvant alone — B. garinii (OspA 10/10  8/10 serotype 6) strain Ma) I Protection against needle challenge with serotype 6 OspA borrelia by improved heterodimer combination vaccine (decreasing doses: 3 μg, 0.3 μg and 0.03 μg) Experiment 16: Experiment 17: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA6-His (SEQ ID NO: 39) 3 x 1.0 μg B. garinii (OspA  4/10 (0.0108)  8/10 (n.s.) serotype 6) strain Ma) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 1.0 μg B. garinii (OspA  0/10 (<0.0001)  0/10 (0.0001) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 1.0 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 1.0 μg Ma) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.1 μg B. garinii (OspA  2/10 (0.0007)  2/10 (0.0054) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.1 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.1 μg Ma) Improved heterodimer combination vaccine: Lip-S1D1-S2D1 (Seq ID No: 29) 3 × 0.01 μg B. garinii (OspA  3/10 (0.0031)  4/10 (n.s.) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 × 0.01 μg serotype 6) strain Lip-S5D1-S6D1 (Seq ID No: 33) 3 × 0.01 μg Ma) Al(OH).sub.3 adjuvant alone — B. garinii (OspA 10/10  9/10 serotype 6) strain Ma) P-value; Fisher's exact test, two-tailed, as compared to the adjuvant alone group, are indicated in parenthesis. not significant (n.s.).

TABLE-US-00007 SEQUENCES SEQ ID NO: 1 S3hybD1: hybrid OspA C-terminal fragment; amino acids of positions 125-176 from Borrelia valaisiana, strain VS116, and amino acids 177-274 from Borrelia garinii, strain PBr, with disulfide bond type 1 and T in position 233 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNY NRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 2 B. valaisiana (strain VS116), OspA aa 125-176 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGK SEQ ID NO: 3 B. garinii (strain PBr, serotype 3), OspA aa 177-274, with T in position 233, from full-length OspA (SEQ ID NO: 8) TKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFT KENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 4 B. valaisiana (strain VS116), OspA MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLVATVDKV ELKGTSDKNNGSGTLEGVKDDKSKVKLTISDDLGETKLETFKEDGTLVSRKVNFKDKSFTEE KFNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITEGT VTLSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQS YNPAGNKLEGTAVEIKTLQELKNALK SEQ ID NO: 5 B. burgdorferi s.s. (strain B31, OspA serotype 1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLE LKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTL SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS NGTKLEGSAVEITKLDEIKNALK SEQ ID NO: 6 B. afzelii (strain K78; OspA serotype 2) MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIE LKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDE MFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT VTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQ KYDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 7 B. garinii (strain PBr, OspA serotype 3) with P in position 233 (embl accession X80256.1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKL ELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEE KFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGT VTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQN YNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 8 B. garinii (strain PBr, OspA serotype 3) with T in position 233 (embl accession ACL34827.1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKL ELKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEE KFNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGT VTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQN YNRAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 9 B. bavariensis (strain PBi, OspA serotype 4) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKL ELKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEK FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVL SKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSA GTNLEGNAVEIKTLDELKNALK SEQ ID NO: 10 B. garinii (strain PHei, OspA serotype 5) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKL ELKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEE KFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVT LSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDS AGTNLEGKAVEITTLKELKNALK SEQ ID NO: 11 B. garinii (strain DK29, OspA serotype 6) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLE LKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEK FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYD SAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 12 B. garinii (strain T25, OspA serotype 7) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLE LKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEE KFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGT VTLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKY NTAGTKLEGSPAEIKDLEALKAALK SEQ ID NO: 13 Borrelia OspA lipidation signal MKKYLLGIGLILALIA SEQ ID NO: 14 Borrelia OspB lipidation signal MRLLIGFALALALIG SEQ ID NO: 15 E. coli lpp lipidation signal MKATKLVLGAVILGSTLLAG SEQ ID NO: 16 LN1 peptide linker constructed from two separate loop regions of the N-terminal half of OspA from B. burgdorferi s.s. strain B31 (aa 65-74 and aa 42-53, amino acid exchange at position 53: D53S) GTSDKNNGSGSKEKNKDGKYS SEQ ID NO: 17 hLFA-1-like sequence from B. burgdorferi s.s. strain B31 (OspA serotype 1) GYVLEGTLTAE SEQ ID NO: 18 Non-hLFA-1-like sequence from B. afzelii strain K78 (OspA serotype 2) NFTLEGKVAND SEQ ID NO: 19 B. burgdorferi s.s. (strain B31, serotype 1), OspA aa 126-273 with replaced hLFA-like sequence from serotype 1 OspA FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLS KNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSN GTKLEGSAVEITKLDEIKNALK SEQ ID NO: 20 B. afzelii (strain K78, serotype 2), OspA aa 126-273 FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTV TLSKELAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQK YDSAGTNLEGTAVEIKTLDELKNALK SEQ ID NO: 21 B. garinii (strain PBr, serotype 3), OspA aa 126-274 FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYN RAGNALEGSPAEIKDLAELKAALK SEQ ID NO: 22 B. bavariensis (strain PBi, serotype 4), OspA aa 126-273 FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVL SKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSA GTNLEGNAVEIKTLDELKNALK SEQ ID NO: 23 B. garinii (strain PHei, serotype 5), OspA aa 126-273 FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTL SKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSA GTNLEGKAVEITTLKELKNALK SEQ ID NO: 24 B. garinii (strain DK29, serotype 6), OspA aa 126-274 FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYD SAGTNLEGKAVEITTLKELKNALK SEQ ID NO: 25 B. garinii (strain T25, serotype 7) OspA aa 126-274 FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVT LSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNT AGTKLEGSPAEIKDLEALKAALK SEQ ID NO: 26 Lip-S4D1-S3hybD1-nt Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 4 and OspA serotype 3 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 linker sequence, serotype 3 OspA fragment comprising amino acids 125-176 of B. valaisiana, strain VS116 (SEQ ID NO: 2) and amino acids 177-274 of B. garinii, strain PBr, serotype 3 (SEQ ID NO: 3) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGG CACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCC TGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACG TGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTG AACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTC CACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAG ATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTC GAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAA TGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGC GAAGTGAGCGAAAAAATTCTGACCCGTAGCAATGGCACCACCCTGGAATATAGCCAGAT GACCGATGCAGAAAATGCAACCAAAGCAGTTGAAACCCTGAAAAACGGTATTAAACTGC CTGGTAATCTGGTTGGTGGTAAAACCAAACTGACCGTTACCTGTGGCACCGTTACCCTGA GCAAAAACATTAGCAAAAGCGGTGAAATTACCGTGGCACTGAATGATACCGAAACCACA CCGGCAGACAAAAAAACCGGTGAATGGAAAAGCGATACCAGCACCCTGACCATTAGTAA AAATAGCCAGAAAACAAAACAGCTGGTGTTTACCAAAGAAAACACCATTACCGTGCAGA ATTATAACCGTGCAGGTAATGCACTGGAAGGTAGTCCGGCAGAAATTAAAGATCTGGCA GAACTGTGTGCAGCCCTGAAATAA SEQ ID NO: 27 Lip-S4D1-S3hybD1-aa: Heterodimer fusion protein of OspA serotype 4 and OspA serotype 3, comprising amino acids 125-176 of B. valaisiana, strain VS116 (SEQ ID NO: 2) and amino acids 177-274 of B. garinii, strain PBr, serotype 3 (SEQ ID NO: 3), with disulfide bond type 1, N-terminalCSS for addition of lipids, LN1 linker sequence, N-terminal lipidation LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTC GTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQ KYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKILTR SNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTVTLSKNISKSGEITV ALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEI KDLAELCAALK SEQ ID NO: 28 Lip-S1D1-S2D1-nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, E. coli lpp lipidation signal, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGG CACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTC TGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAA TGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACT GAATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCT CGACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAA AACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGT GGAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACA ATGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGG CGAACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAA ATGAAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGG CAAAGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAA AAGAAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAA GCGACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAA TAGCAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAAT ACGACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAA CTGTGTAACGCCCTGAAA SEQ ID NO: 29 Lip-S1D1-S2D1-aa: Heterodimer fusion protein of OspA serotype 1 and OspA serotype 2 with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, aa 164-174 of OspA serotype 1 replaced by non-hLFA-1-like sequence NFTLEGKVAND, N-terminal lipidation LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKC GTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITV QQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTM TRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVT VALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAV EIKTLDELCNALK SEQ ID NO: 30 Lip-S4D1-S3D1-nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 4 and 3 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGG CACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCC TGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACG TGCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTG AACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTC CACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAG ATACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTC GAAATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAA TGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCA AACTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATC AAAAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTAC CCTGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTA AAAACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCG GCTGACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAA CTCGCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACT ATAATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAA CTGTGTGCGGCACTGAAA SEQ ID NO: 31 Lip-S4D1-S3D1-aa: Heterodimer fusion protein of OspA serotypes 4 and 3 both with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidation LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTC GTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQ KYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVT RANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVA LNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIK DLAELCAALK SEQ ID NO: 32 Lip-S5D1-S6D1-nt: Coding sequence for intermediate and final heterodimer fusion proteins of OspA serotypes 6 both with disulfide bond type 1, E. coli lpp lipidation signal, N-terminal CSS for addition of lipids, LN1 linker sequence ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGG CACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTC TGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTG ACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCC CTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTC GACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAG ATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTG GAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAA TGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTG AAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATT AAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTAC CCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAA AAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGT GCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAA CTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCT ATGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAA CTGTGTAATGCTCTGAAA SEQ ID NO: 33 Lip-S5D1-S6D1-aa: Heterodimer fusion protein of OspA serotypes 6 both with disulfide bond type 1, N-terminal CSS for addition of lipids, LN1 linker sequence, N-terminal lipidation LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTC GTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQ NYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVR ANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAA LDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEI TTLKELCNALK SEQ ID NO: 34 B. burgdorferi (strain B31, OspA serotype 1) aa 18-273, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminal CSSF for addition of lipids CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSG VLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKII TRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSV ELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEI TKLDEIKNALKLEHHHHHH SEQ ID NO: 35 B. afzelii (strain K78; OspA serotype 2) aa 18-273, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminal CSSF for addition of lipids CSSFKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSG VLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKT MTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEV TVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTA VEIKTLDELKNALKLEHHHHHH SEQ ID NO: 36 B. garinii (strain PBr; OspA serotype 3) aa 18-274, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminal CSSF for addition of lipids CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSG VLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKV VTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEIT VALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPA EIKDLAELKAALKLEHHHHHH SEQ ID NO: 37 B. bavariensis (strain PBi; OspA serotype 4) aa 18-273, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminal CSSF for addition of lipids CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSG TLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILR ANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELN DSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTL DELKNALKLEHHHHHH SEQ ID NO: 38 B. garinii (strain PHei; OspA serotype 5) aa 18-273, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH, SEQ ID NO: 64), N-terminal CSSF (SEQ ID NO: 65) for addition of lipids CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSG TLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIV RANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVA LDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITT LKELKNALKLEHHHHHH SEQ ID NO: 39 B. garinii (strain DK29; OspA serotype 6) aa 18-274, lpp lipidation signal sequence removed (MKATKLVLGAVILGSTLLAG, SEQ ID NO: 15), C-terminal His tag (LEHHHHHH), N-terminal CSSF for addition of lipids CSSFKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSG TLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTI VRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEIT AALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAV EITTLKELKNALKLEHHHHHH SEQ ID NO: 40 Chimeric OspA Serotype1/Serotype2, N-terminal lipidation, His-tagged, including the OspB lipidation signal sequence: MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKG TSDKNNGSGVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFN EKGEVSEKIITMADGTRLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSM NISKSGEVSVELNDTDSSAATKKTAAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAG TNLEGTAVEIKTLDELKNALKLEHHHHHH SEQ ID NO: 41 Chimeric OspA Serotype5/Serotype3, N-terminal lipidation, His-tagged, including the OspB lipidation signal sequence: MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMKVLVSKEKDKNGKYSLMATVEKLELK GTSDKNNGSGTLEGEKTNKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFN EKGEISEKTIVMANGTRLEYTDIKSDKTGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVTLS MNISKSGEITVALDDTDSSGNKKSGTWDSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAG NALEGSPAEIKDLAELKAALKLEHHHHHH SEQ ID NO: 42 Chimeric OspA Serotype6/Serotype4, N-terminal lipidation, His-tagged, including the OspB lipidation signal sequence: MRLLIGFALALALIG (SEQ ID NO: 14) which is cleaved during processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMTVLVSKEKDKNGKYSLEATVDKLELKG TSDKNNGSGTLEGEKTNKSKVKLTIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTEEKFNE KGETSEKTIVMANGTRLEYTDIKSDGSGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVVLS MNILKSGEITVALDDSDTTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSA GTNLEGNAVEIKTLDELKNALKLEHHHHHH SEQ ID NO: 43 S1D1 FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTL SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS NGTKLEGSAVEITKLDEICNALK SEQ ID NO: 44 S2D1 FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTV TLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQK YDSAGTNLEGTAVEIKTLDELCNALK SEQ ID NO: 45 S3D1 FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNY NRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 46 S4D1 FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVL SKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSA GTNLEGNAVEIKTLDELCNALK SEQ ID NO: 47 S5D1 FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTL SKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSA GTNLEGKAVEITTLKELCNALK SEQ ID NO: 48 S6D1 FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYD SAGTNLEGKAVEITTLKELCNALK SEQ ID NO: 49 S3HYBD1 (BVA) FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNY NRAGNALEGSPAEIKDLAELCAALK SEQ ID NO: 50 BVAD1 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITCGTVT LSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQSYNP AGNKLEGTAVEIKTLQELCNALK SEQ ID NO: 51 S3hybD1(Bsp): hybrid OspA C-terminal fragment; amino acids 126-175 from Borrelia spielmanii and amino acids 177-274 from Borrelia garinii, strain PBr, with disulfide bond type 1 and T in position 233 FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKTKLTVTCGTVTL SKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNR AGNALEGSPAEIKDLAELCAALK SEQ ID NO: 52 MSPD1 FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKATLTVKCGTVTL SKNIDKSGEVTVALNDTDSTAATKKTGAWDSKTSTLTITVNSKKTKDLVFTKQDTITVQKYD SAGTTLEGSAVEIKTLDELCNALK SEQ ID NO: 53 Forward primer for the 16S-23S intergenic spacer GTATGTTTAGTGAGGGGGGTG SEQ ID NO: 54 Reverse primer for the 16S-23S intergenic spacer GGATCATAGCTCAGGTGGTTAG SEQ ID NO: 55 Forward nested primer for the 16S-23S intergenic spacer AGGGGGGTGAAGTCGTAACAAG SEQ ID NO: 56 Reverse nested primer for the 16S-23S intergenic spacer GTCTGATAAACCTGAGGTCGGA SEQ ID NO: 57 Forward primer for the RecA gene of Borrelia CATGCTCTTGATCCTGTTTA SEQ ID NO: 58 Reverse primer for the RecA gene of Borrelia CCCATTTCTCCATCTATCTC SEQ ID NO: 59 25-mer peptide from the Invariable Region 6 (IR6) of VlSE MKKDDQIAAAMVLRGMAKDGQFALK SEQ ID NO: 60 Mouse cathelin RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE SEQ ID NO: 61 KLK peptide KLKLLLLLKLK SEQ ID NO: 62 N-terminal peptide for lipidation CKQN SEQ ID NO: 63 5′-(dIdC).sub.13-3′ dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC dIdC

[0312] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.