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. A polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 27.

2. A pharmaceutical composition comprising the polypeptide according to claim 1, and optionally a pharmaceutically acceptable carrier or excipient.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutically acceptable excipient comprises L-methionine and/or aluminium hydroxide.

4. A pharmaceutical composition comprising the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 27 and SEQ ID NO: 33.

5. The polypeptide according to claim 1 for use in a method of treating or protecting against a Borrelia infection, wherein the Borrelia infection is a B. burgdorferi s.s. or B. afzelii infection.

6. The pharmaceutical composition according to claim 2 for use in a method of treating or protecting against a Borrelia infection, wherein the Borrelia infection is a B. burgdorferi s.s. or B. afzelii infection.

7. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is a vaccine.

8. The pharmaceutical composition according to claim 4, further comprising a pharmaceutically acceptable carrier or excipient.

9. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable excipient comprises L-methionine and/or aluminium hydroxide.

10. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is a vaccine.

11. The pharmaceutical composition according to claim 4 for use in a method of treating or protecting against a Borrelia infection, wherein the Borrelia infection is B. burgdorferi s.s. or B. afzelii infection.

Description

(1) In connection with the present invention

(2) 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).

(3) FIG. 2 shows the amino acid alignment of Lip-S4D1-S3hybD1, the mutant serotype 3 OspA fusion fragment-containing heterodimer protein of the invention, with the heterodimer protein Lip-S4D1-S3D1.

(4) FIG. 3 schematically shows the production of mutant OspA fragment heterodimers according to the current invention.

(5) 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.

(6) 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.

(7) 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.

(8) 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.

(9) Table 1 compares the purification yield of the Lip-S4D1-S3hybD1 heterodimer and the Lip-S4D1-S3D1 heterodimer

(10) 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.

(11) The figures and tables which may be referred to in the specification are described below in more detail.

(12) 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.

(13) FIG. 2 Amino acid sequence alignment of Lip-S4D1-S3hybD1 and Lip-S4D1-S3D1 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.

(14) 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.

(15) 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.

(16) 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.)

(17) FIG. 6 Antibody titers generated to all six serotypes of full-length OspA proteins. Mice were immunized three times with 3 ?g each of the indicated combination vaccines: Lip-S1D1-S2D1, Lip-S4D1-S3D1 and Lip-S5D1-S6D1 together in a 1:1:1 ratio (Het combo); Lip-S1D1-S2D1, Lip-S4D1-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.

(18) 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

(19) Motivation to Construct Hybrid OspA ST3 C-Terminal Fragments

(20) 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.

(21) 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.

(22) Molecular Modelling

(23) 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, Schr?dinger LLC).

(24) 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.

(25) 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).

(26) Application in the Form of Heterodimers Containing the Hybrid OspA Fragment

(27) 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, S4D1-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.

(28) 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

(29) Cloning and Expression of Lipidated Non-his-Tagged Mutant OspA Fragment Heterodimers

(30) 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.

(31) Purification of Lipidated Mutant OspA Fragment Heterodimers

(32) 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.

(33) Formulation of the Combination Vaccine

(34) 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 alumninium hydroxide and antigen, as described in our previous application WO2014/006226. An optimal formulation of 40 ?g/mL each of three heterodimers (120 ?g 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.

(35) Results

(36) 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.)

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

Example 3

Immunogenicity of Lipidated Mutant OspA Fragment Heterodimers of Different Serotypes

(38) Immunization of Mice

(39) Female C3H/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 ?L each were administered at two week intervals. Each dose contained 1 ?g 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-S4D1-S3D1 (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.

(40) OspA ELISA

(41) ELISA plates (Maxisorp, Nunc, Denmark) were coated with 50 ng (1 ?g/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 ?L 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 ?L (overflow) PBST (0.1% Tween-20, PBS). Five-fold dilutions of the sera were made in blocking buffer and 50 ?L were added to each well and incubated for 1 hour at ambient temperature. Plates were washed three times with 300 ?L (overflow) PBST. The secondary antibody (horseradish peroxidase [HRP]-conjugated rabbit anti-mouse IgG, DAKO, Denmark) was diluted 1:2000 in blocking buffer and 50 ?L was added to each well and incubated for 1 hour at ambient temperature. Plates were washed three times with 300 ?L (overflow) PBST. ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Sigma-Aldrich, USA) was used as substrate for HRP, 50 ?L 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 ?L 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.

(42) Flow Cytometry

(43) 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 ?L 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 ?m, Corning, USA). Spirochetes were dissolved in 100 ?L 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 ?L HBSS-B and then resuspended in 100 ?L 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 ?L at HBSS-B and then resuspended in 200 ?L at HBSS containing 2.5 ?M 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 ?L HBSS. Labelled spirochetes were measured with an FC500 (Beckman Coulter) flow cytometer, gated for SYTO-17 positive events.

(44) Results

(45) 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).

(46) 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.

(47) 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

(48) Immunization of Mice

(49) Female C3H/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 ?L 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.

(50) Needle Challenge of Immunized Mice with In Vitro Grown Borrelia

(51) Two weeks after the last immunization, mice were challenged s.c. with spirochetes diluted in 100 ?L 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.

(52) Challenge of Immunized Mice with Ticks Infected with B. burgdorferi or B. afzelii (Tick Challenge)

(53) 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 Pra1 expressing OspA serotype 1 (experiments 4 and 5) or B. afzelii expressing OspA serotype 2 (experimens 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.

(54) Sacrifice of Mice and Collection of Material

(55) 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.

(56) ELISA with the Invariable Region 6 (IR6) of VISE

(57) 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 ?L/well (1 ?g/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 ?L/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 ?L of 1.3 ?g/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 ?L/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.

(58) DNA Extraction and Purification

(59) 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 ?L recombinant Proteinase K (PCR grade; 14-22 mg/mL, Roche). The DNA was eluted in 50 ?L 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.

(60) qPCR Targeting recA

(61) 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 ?L SSoAdvanced? SYBR? Green Supermix, 0.3 ?L of each primer (10 ?M), and 7.4 ?L water was prepared for each experiment. Eighteen ?L of master mix was mixed with 2 ?L 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.

(62) Western Blot

(63) Binding of final sera to whole cell lysates from borrelia belonging to the corresponding OspA serotype was analyzed by western blot. Briefly, 2.5 ?g 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).

(64) Infection Readout

(65) 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).

(66) Results

(67) 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.

(68) 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.

(69) The improved heterodimer combination vaccine conferred significant protection (p-values <0.05), at a 3 ?g 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 ?g 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.

(70) 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).

(71) 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 ?g) 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 ?g B. burgdorferi 0/10 (<0.0001) 1/10 (<0.0001) (OspA serotype 1) strain ZS7 Chimera combination vaccine: B. burgdorferi 0/10 (<0.0001) 0/10 (<0.0001) Lip-Chimeric OspA ST1/ST2-His 3 ? 1.0 ?g (OspA serotype 1) (Seq ID No: 40) strain ZS7 Lip-Chimeric OspA ST5/ST3-His 3 ? 1.0 ?g (Seq ID No: 41) Lip-Chimeric OspA ST6/ST4-His 3 ? 1.0 ?g (Seq ID No: 42) Improved heterodimer combination B. burgdorferi 0/10 (<0.0001) 0/10 (<0.0001) vaccine: (OspA serotype 1) Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g strain ZS7 Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g Al(OH).sub.3 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 ?g) 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 ?g 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: Tick challenge with 0/8 (0.0060) 0/7 (0.0093) Lip-Chimeric OspA ST1/ST2-His 3 ? 1.0 ?g B. burgdorferi (Seq ID No: 40) (OspA serotype 1) Lip-Chimeric OspA ST5/ST3-His 3 ? 1.0 ?g strain Pra4 (Exp. 3) (Seq ID No: 41) or Pra1 (Exp. 4) Lip-Chimeric OspA ST6/ST4-His 3 ? 1.0 ?g (Seq ID No: 42) Improved heterodimer combination Tick challenge with 0/5 (0.0260) 0/7 (0.0093) vaccine: B. burgdorferi Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g (OspA serotype 1) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g strain Pra4 (Exp. 3) Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g 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 ?g and 0.3 ?g) Experiment 5: Infected/Total Immunogen Dose Challenge (p-value) Improved heterodimer combination Tick challenge with 1/7 (0.0174) vaccine: B. burgdorferi Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g (OspA serotype 1) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g strain Pra1 Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g Improved heterodimer combination Tick challenge with 1/9 (0.0059) vaccine: B. burgdorferi Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.1 ?g (OspA serotype 1) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.1 ?g strain Pra1 Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.1 ?g Al(OH).sub.3 adjuvant alone Tick challenge with 7/8 B. burgdorferi (OspA serotype 1) strain Pra1 D Protection against tick challenge with serotype 2 OspA borrelia by chimera combination vaccine and improved heterodimer combination vaccine (one dose: 3 ?g) 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 ?g Tick challenge with 0/8 (0.0016) 0/9 (0.0002) B. afzelii (OspA serotype 2) strain IS1 Chimera combination vaccine: Tick challenge with 0/7 (0.0025) 0/9 (0.0002) Lip-Chimeric OspA ST1/ST2-His 3 ? 1.0 ?g B. afzelii (OspA (Seq ID No: 40) serotype 2) strain Lip-Chimeric OspA ST5/ST3-His 3 ? 1.0 ?g IS1 (Seq ID No: 41) Lip-Chimeric OspA ST6/ST4-His 3 ? 1.0 ?g (Seq ID No: 42) Improved heterodimer combination Tick challenge with 0/9 (0.0010) 0/7 (0.0003) vaccine: B. afzelii (OspA Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g serotype 2) strain Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g IS1 Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g 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 ?g and 0.003 ?g) Experiment 8: Experiment 9: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Improved heterodimer combination Tick challenge with 0/9 (0.0004) 2/7 (0.0096) vaccine: B. afzelii (OspA Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.01 ?g serotype 2) strain Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.01 ?g IS1 Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.01 ?g Improved heterodimer combination Tick challenge with 7/10 (n.s.) 1/8 (0.0008) vaccine: B. afzelii (OspA Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.001 ?g serotype 2) strain Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.001 ?g IS1 Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.001 ?g 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 ?g) 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 ?g B. garinii (OspA 0/10 (<0.0001) 0/10 (<0.0001) serotype 5) strain PHei) Improved heterodimer combination B. garinii (OspA 0/10 (<0.0001) 1/10 (<0.0001) vaccine: serotype 5) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g PHei) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g 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 ?g, 0.3 ?g and 0.03 ?g) 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 ?g B. garinii (OspA 0/10 (0.0007) 0/10 (<0.001) serotype 5) strain PHei) Improved heterodimer combination B. garinii (OspA 0/10 (<0.0007) 1/10 (0.0002) vaccine: serotype 5) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g PHei) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g Improved heterodimer combination B. garinii (OspA 0/10 (<0.0007) 0/10 (<0.0001) vaccine: serotype 5) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.1 ?g PHei) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.1 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.1 ?g Improved heterodimer combination B. garinii (OspA 2/10 (<0.0219) 3/10 (0.0047) vaccine: serotype 5) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.01 ?g PHei) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.01 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.01 ?g 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 ?g) 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 ?g B. garinii (OspA 4/10 (0.0108) 6/10 (n.s.).sup. serotype 6) strain Ma) Improved heterodimer combination B. garinii (OspA 0/10 (<0.0001) 0/10 (0.0007) vaccine: serotype 6) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g Ma) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g 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 ?g, 0.3 ?g and 0.03 ?g) Experiment 16: Experiment 17: Infected/Total Infected/Total Immunogen Dose Challenge (p-value) (p-value) Lip-OspA6-His (SEQ ID NO: 39) 3 ? 1.0 ?g B. garinii (OspA 4/10 (0.0108) 8/10 (n.s.).sup. serotype 6) strain Ma) Improved heterodimer combination B. garinii (OspA 0/10 (<0.0001) 0/10 (0.0001) vaccine: serotype 6) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 1.0 ?g Ma) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 1.0 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 1.0 ?g Improved heterodimer combination B. garinii (OspA 2/10 (0.0007) 2/10 (0.0054) vaccine: serotype 6) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.1 ?g Ma) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.1 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.1 ?g Improved heterodimer combination B. garinii (OspA 3/10 (0.0031) 4/10 (n.s.).sup. vaccine: serotype 6) strain Lip-S1D1-S2D1 (Seq ID No: 29) 3 ? 0.01 ?g Ma) Lip-S4D1-S3hybD1 (Seq ID No: 27) 3 ? 0.01 ?g Lip-S5D1-S6D1 (Seq ID No: 33) 3 ? 0.01 ?g 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.).

(72) TABLE-US-00007 SEQUENCES SEQIDNO:1 S3hybD1:hybridOspAC-terminalfragment;aminoacids ofpositions125-176fromBorreliavalaisiana,strain VS116,andaminoacids177-274fromBorreliagarinii, strainPBr,withdisulfidebondtype1andTin position233 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYN RAGNALEGSPAEIKDLAELCAALK SEQIDNO:2 B.valaisiana(strainVS116),OspAaa125-176 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGK SEQIDNO:3 B.garinii(strainPBr,serotype3),OspAaa177-274, withTinposition233,fromfull-lengthOspA (SEQIDNO:8) TKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTK ENTITVQNYNRAGNALEGSPAEIKDLAELCAALK SEQIDNO:4 B.valaisiana(strainVS116),OspA MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLVATVDKVE LKGTSDKNNGSGTLEGVKDDKSKVKLTISDDLGETKLETFKEDGTLVSRKVNFKDKSFTEEK FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITEGTVT LSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQSYNP AGNKLEGTAVEIKTLQELKNALK SEQIDNO:5 B.burgdorferis.s.(strainB31,OspAserotype1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLE LKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTL SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS NGTKLEGSAVEITKLDEIKNALK SEQIDNO:6 B.afzelii(strainK78;OspAserotype2) MKKYLLGIGLILALIACKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIE LKGTSDKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDE MFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGT VTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQK YDSAGTNLEGTAVEIKTLDELKNALK SEQIDNO:7 B.garinii(strainPBr,OspAserotype3)withPin position233(emblaccessionX80256.1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLE LKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEK FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVT LSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNR AGNALEGSPAEIKDLAELKAALK SEQIDNO:8 B.garinii(strainPBr,OspAserotype3)withTin position233(emblaccessionACL34827.1) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLE LKGTSDKSNGSGVLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEK FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVT LSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNR AGNALEGSPAEIKDLAELKAALK SEQIDNO:9 B.bavariensis(strainPBi,OspAserotype4) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLE LKGTSDKSNGSGTLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKF NAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLS KHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAG TNLEGNAVEIKTLDELKNALK SEQIDNO:10 B.garinii(strainPHei,OspAserotype5) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLE LKGTSDKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEK FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTL SKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAG TNLEGKAVEITTLKELKNALK SEQIDNO:11 B.garinii(strainDK29,OspAserotype6) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLE LKGTSDKNNGSGTLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEK FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDS AGTNLEGKAVEITTLKELKNALK SEQIDNO:12 B.garinii(strainT25,OspAserotype7) MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLEATVDKLE LKGTSDKNNGSGVLEGVKAAKSKAKLTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEE KFNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTV TLSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYN TAGTKLEGSPAEIKDLEALKAALK SEQIDNO:13 BorreliaOspAlipidationsignal MKKYLLGIGLILALIA SEQIDNO:14 BorreliaOspBlipidationsignal MRLLIGFALALALIG SEQIDNO:15 E.coli1pplipidationsignal MKATKLVLGAVILGSTLLAG SEQIDNO:16 LN1peptidelinkerconstructedfromtwoseparateloopregions oftheN-terminalhalfofOspAfromB.burgdorferis.s.strain B31(aa65-74andaa42-53,aminoacidexchangeatposition 53:D53S) GTSDKNNGSGSKEKNKDGKYS SEQIDNO:17 hLFA-1-likesequencefromB.burgdorferis.s.strainB31 (OspAserotype1) GYVLEGTLTAE SEQIDNO:18 Non-hLFA-1-likesequencefromB.afzeliistrainK78 (OspAserotype2) NFTLEGKVAND SEQIDNO:19 B.burgdorferis.s.(strainB31,serotype1),OspAaa126-273 withreplacedhLFA-likesequencefromserotype1OspA FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTLS KNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSN GTKLEGSAVEITKLDEIKNALK SEQIDNO:20 B.afzelii(strainK78,serotype2),OspAaa126-273 FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVT LSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYD SAGTNLEGTAVEIKTLDELKNALK SEQIDNO:21 B.garinii(strainPBr,serotype3),OspAaa126-274 FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVT LSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNR AGNALEGSPAEIKDLAELKAALK SEQIDNO:22 B.bavariensis(strainPBi,serotype4),OspAaa126-273 FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLS KHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAG TNLEGNAVEIKTLDELKNALK SEQIDNO:23 B.garinii(strainPHei,serotype5),OspAaa126-273 FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTL SKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAG TNLEGKAVEITTLKELKNALK SEQIDNO:24 B.garinii(strainDK29,serotype6),OspAaa126-274 FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDS AGTNLEGKAVEITTLKELKNALK SEQIDNO:25 B.garinii(strainT25,serotype7)OspAaa126-274 FNDKGKLSEKVVTRANGTRLEYTEIQNDGSGKAKEVLKSLTLEGTLTADGETKLTVEAGTVT LSKNISESGEITVELKDTETTPADKKSGTWDSKTSTLTISKNSQKTKQLVFTKENTITVQKYNT AGTKLEGSPAEIKDLEALKAALK SEQIDNO:26 Lip-S4D1-S3hybD1-ntCodingsequenceforintermediateand finalheterodimerfusionproteinsofOspAserotype4and OspAserotype3withdisulfidebondtype1,E.coli1pp lipidationsignal,LN1linkersequence,serotype3OspA fragmentcomprisingaminoacids125-176ofB.valaisiana, strainVS116(SEQIDNO:2)andaminoacids177-274ofB. garinii,strainPBr,serotype3 (SEQIDNO:3) ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGG CACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCC TGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGT GCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGA ACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCC ACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGAT ACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGA AATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATG GCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCGAA GTGAGCGAAAAAATTCTGACCCGTAGCAATGGCACCACCCTGGAATATAGCCAGATGAC CGATGCAGAAAATGCAACCAAAGCAGTTGAAACCCTGAAAAACGGTATTAAACTGCCTG GTAATCTGGTTGGTGGTAAAACCAAACTGACCGTTACCTGTGGCACCGTTACCCTGAGCA AAAACATTAGCAAAAGCGGTGAAATTACCGTGGCACTGAATGATACCGAAACCACACCG GCAGACAAAAAAACCGGTGAATGGAAAAGCGATACCAGCACCCTGACCATTAGTAAAAA TAGCCAGAAAACAAAACAGCTGGTGTTTACCAAAGAAAACACCATTACCGTGCAGAATT ATAACCGTGCAGGTAATGCACTGGAAGGTAGTCCGGCAGAAATTAAAGATCTGGCAGAA CTGTGTGCAGCCCTGAAATAA SEQIDNO:27 Lip-S4D1-S3hybD1-aa:HeterodimerfusionproteinofOspA serotype4andOspAserotype3,comprisingaminoacids 125-176ofB.valaisiana,strainVS116(SEQIDNO:2) andaminoacids177-274ofB.garinii,strainPBr, serotype3(SEQIDNO:3),withdisulfidebondtype1, N-terminalCSSforadditionoflipids,LN1linkerse- quence,N-terminallipidationLip CSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCG TVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQK YDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNEKGEVSEKILTRS NGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTVTLSKNISKSGEITVAL NDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKD LAELCAALK SEQIDNO:28 Lip-S1D1-S2D1-nt:Codingsequenceforintermediateand finalheterodimerfusionproteinsofOspAserotype1and OspAserotype2withdisulfidebondtype1,E.coli1pp lipidationsignal,LN1linkersequence,aa164-174of OspAserotype1replacedbynon-hLFA-1-likesequence NFTLEGKVAND ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAACGAAAAGGGCGAAGTCAGCGAAAAAATCATTACCCGCGCAGACGG CACCCGCCTGGAATACACCGGCATCAAATCGGACGGCAGCGGCAAAGCGAAAGAAGTTC TGAAAAACTTTACCCTGGAAGGCAAAGTCGCAAATGATAAAACCACCCTGGTGGTGAAA TGCGGCACCGTTACGCTGAGCAAAAACATTAGTAAATCCGGTGAAGTCTCTGTGGAACTG AATGATACCGACAGCTCTGCGGCCACCAAGAAAACCGCAGCTTGGAACTCAGGCACCTC GACGCTGACCATTACGGTTAATAGCAAGAAAACCAAAGATCTGGTCTTCACGAAAGAAA ACACCATCACGGTGCAGCAATATGACAGCAATGGTACCAAACTGGAAGGCTCCGCTGTG GAAATCACGAAACTGGATGAAATCTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAA TGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGAAAAAGGCG AACTGTCGGCGAAAACGATGACGCGTGAAAACGGCACCAAACTGGAATATACGGAAATG AAAAGCGATGGCACCGGTAAAGCGAAAGAAGTTCTGAAAAACTTTACCCTGGAAGGCAA AGTCGCCAATGACAAAGTCACCCTGGAAGTGAAATGCGGCACCGTTACGCTGTCAAAAG AAATTGCAAAATCGGGTGAAGTGACCGTTGCTCTGAACGATACGAATACCACGCAAGCG ACCAAGAAAACCGGCGCCTGGGACAGCAAAACCTCTACGCTGACCATTAGTGTTAATAG CAAGAAAACCACGCAGCTGGTCTTCACCAAACAAGATACGATCACCGTGCAGAAATACG ACAGTGCGGGTACCAACCTGGAAGGCACGGCTGTTGAAATCAAAACCCTGGACGAACTG TGTAACGCCCTGAAA SEQIDNO:29 Lip-S1D1-S2D1-aa:HeterodimerfusionproteinofOspA serotype1andOspAserotype2withdisulfidebond type1,N-terminalCSSforadditionoflipids,LN1 linkersequence,aa164-174ofOspAserotype1re- placedbynon-hLFA-1-likesequenceNFTLEGKVAND,N- terminallipidation LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCG TVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQ QYDSNGTKLEGSAVEITKLDEICNALKGTSDKNNGSGSKEKNKDGKYSFNEKGELSAKTMTR ENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVA LNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIK TLDELCNALK SEQIDNO:30 Lip-S4D1-S3D1-nt:Codingsequenceforintermediateand finalheterodimerfusionproteinsofOspAserotypes4 and3bothwithdisulfidebondtype1,E.coli1pp lipidationsignal,N-terminalCSSforadditionof lipids,LN1linkersequence ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAATGCTAAGGGCGAACTGAGCGAAAAAACGATCCTGCGTGCGAATGG CACCCGTCTGGAATACACCGAAATCAAATCCGATGGTACGGGCAAAGCAAAGGAAGTCC TGAAAGATTTTGCTCTGGAAGGTACCCTGGCGGCCGACAAAACCACGCTGAAGGTGACGT GCGGCACCGTGGTTCTGAGCAAACATATTCCGAACTCTGGTGAAATCACCGTTGAACTGA ACGATAGCAATTCTACGCAGGCAACCAAAAAGACGGGCAAATGGGACAGTAATACCTCC ACGCTGACCATTTCAGTCAACTCGAAAAAGACCAAAAATATTGTGTTCACGAAGGAAGAT ACGATCACCGTTCAAAAATATGACTCCGCGGGCACCAACCTGGAAGGCAATGCCGTCGA AATCAAAACCCTGGATGAACTGTGTAACGCCCTGAAGGGTACTAGTGACAAAAACAATG GCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTTAACGATAAGGGCAAA CTGTCGGAAAAAGTGGTCACCCGCGCAAATGGCACCCGCCTGGAATACACGGAAATCAA AAACGATGGTAGCGGCAAAGCGAAGGAAGTTCTGAAAGGCTTTGCCCTGGAAGGTACCC TGACGGATGGCGGTGAAACCAAACTGACCGTGACGTGCGGCACCGTTACGCTGTCTAAA AACATTAGCAAGTCTGGTGAAATCACGGTCGCACTGAATGATACCGAAACCACGCCGGCT GACAAAAAGACCGGCGAATGGAAAAGTGACACCTCCACGCTGACCATTTCAAAGAACTC GCAGAAACCGAAGCAACTGGTCTTCACCAAAGAAAACACGATCACCGTGCAGAACTATA ATCGTGCCGGTAATGCTCTGGAAGGCTCACCGGCTGAAATCAAGGACCTGGCTGAACTGT GTGCGGCACTGAAA SEQIDNO:31 Lip-S4D1-S3D1-aa:HeterodimerfusionproteinofOspA serotypes4and3bothwithdisulfidebondtype1, N-terminalCSSforadditionoflipids,LN1linker sequence,N-terminallipidation LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCG TVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQK YDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNGSGSKEKNKDGKYSFNDKGKLSEKVVTR ANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVTLSKNISKSGEITVAL NDTETTPADKKTGEWKSDTSTLTISKNSQKPKQLVFTKENTITVQNYNRAGNALEGSPAEIKD LAELCAALK SEQIDNO:32 Lip-S5D1-S6D1-nt:Codingsequenceforintermediateand finalheterodimerfusionproteinsofOspAserotypes6 bothwithdisulfidebondtype1,E.coli1pplipidation signal,N-terminalCSSforadditionoflipids,LN1 linkersequence ATGAAAGCTACTAAACTGGTACTGGGCGCGGTAATCCTGGGTTCTACTCTGCTGGCAGGT TGCTCAAGCTTCAACGAAAAGGGCGAAATCTCAGAAAAAACCATCGTCCGCGCTAACGG CACCCGCCTGGAATACACCGACATCAAATCAGACAAGACCGGTAAAGCGAAGGAAGTTC TGAAAGATTTTACGCTGGAAGGTACCCTGGCAGCAGACGGTAAAACCACGCTGAAGGTG ACCTGCGGTACCGTTACGCTGTCCAAAAACATTAGTAAGTCCGGCGAAATCACGGTCGCC CTGGATGACACCGATAGCTCTGGCAACAAAAAGAGCGGTACCTGGGATTCAGGCACCTC GACGCTGACCATTTCTAAAAATCGTACGAAAACCAAGCAGCTGGTCTTCACGAAAGAAG ATACGATCACCGTGCAAAACTATGACAGCGCAGGTACCAATCTGGAAGGCAAAGCTGTG GAAATTACCACGCTGAAAGAACTGTGTAATGCTCTGAAAGGTACTAGTGACAAAAACAA TGGCTCTGGTAGCAAAGAGAAAAACAAAGATGGCAAGTACTCATTCAACGGCAAAGGTG AAACGAGCGAAAAGACCATCGTGCGTGCGAACGGTACCCGCCTGGAATATACGGACATT AAATCGGACGGCAGCGGCAAAGCAAAGGAAGTCCTGAAAGATTTTACGCTGGAAGGTAC CCTGGCAGCAGACGGTAAAACCACGCTGAAGGTGACGTGCGGCACCGTGGTTCTGTCAA AAAACATTCTGAAGTCGGGTGAAATCACCGCAGCTCTGGATGACAGCGATACCACGCGT GCTACGAAAAAGACCGGTAAATGGGATAGCAAGACCTCTACGCTGACCATTAGTGTCAA CTCCCAGAAAACGAAGAATCTGGTGTTCACCAAAGAAGATACGATCACCGTTCAACGCTA TGACAGTGCGGGCACCAACCTGGAAGGCAAAGCCGTTGAAATTACCACGCTGAAAGAAC TGTGTAATGCTCTGAAA SEQIDNO:33 Lip-S5D1-S6D1-aa:HeterodimerfusionproteinofOspA serotypes6bothwithdisulfidebondtype1,N-terminal CSSforadditionoflipids,LN1linkersequence,N- terminallipidation LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTC GTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQN YDSAGTNLEGKAVEITTLKELCNALKGTSDKNNGSGSKEKNKDGKYSFNGKGETSEKTIVRA NGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALD DSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTL KELCNALK SEQIDNO:34 B.burgdorferi(strainB31,OspAserotype1)aa18-273,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLELKGTSDKNNGSG VLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFNEKGEVSEKII TRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTLSKNISKSGEVSVE LNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEIT KLDEIKNALKLEHHHHHH SEQIDNO:35 B.afzelii(strainK78;OspAserotype2)aa18-273,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSASVDLPGEMKVLVSKEKDKDGKYSLKATVDKIELKGTSDKDNGSG VLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSSKDKTSTDEMFNEKGELSAKT MTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKEGTVTLSKEIAKSGEV TVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTA VEIKTLDELKNALKLEHHHHHH SEQIDNO:36 B.garinii(strainPBr;OspAserotype3)aa18-274,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKSNGSG VLEGEKADKSKAKLTISQDLNQTTFEIFKEDGKTLVSRKVNSKDKSSTEEKFNDKGKLSEKVV TRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTEGTVTLSKNISKSGEITV ALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEI KDLAELKAALKLEHHHHHH SEQIDNO:37 B.bavariensis(strainPBi;OspAserotype4)aa18-273,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSVSVDLPGEMKVLVSKEKDKDGKYSLMATVDKLELKGTSDKSNGSG TLEGEKSDKSKAKLTISEDLSKTTFEIFKEDGKTLVSKKVNSKDKSSIEEKFNAKGELSEKTILR ANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTEGTVVLSKHIPNSGEITVELN DSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTL DELKNALKLEHHHHHH SEQIDNO:38 B.garinii(strainPHei;OspAserotype5)aa18-273,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSVSVDLPGGMKVLVSKEKDKDGKYSLMATVEKLELKGTSDKNNGSG TLEGEKTDKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNEKGEISEKTIVR ANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVTLSKNISKSGEITVAL DDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTL KELKNALKLEHHHHHH SEQIDNO:39 B.garinii(strainDK29;OspAserotype6)aa18-274,1pp lipidationsignalsequenceremoved(MKATKLVLGAVILGSTLLAG, SEQIDNO:15),C-terminalHistag(LEHHHHHH),N-terminal CSSFforadditionoflipids CSSFKQNVSSLDEKNSVSVDLPGGMTVLVSKEKDKDGKYSLEATVDKLELKGTSDKNNGSG TLEGEKTDKSKVKSTIADDLSQTKFEIFKEDGKTLVSKKVTLKDKSSTEEKFNGKGETSEKTIV RANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTEGTVVLSKNILKSGEITAA LDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEIT TLKELKNALKLEHHHHHH SEQIDNO:40 ChimericOspASerotype1/Serotype2,N-terminallipidation, His-tagged,includingtheOspBlipidationsignalsequence: MRLLIGFALALALIG(SEQIDNO:14)whichiscleavedduring processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGEMKVLVSKEKDKNGKYDLIATVDKLELKG TSDKNNGSGVLEGVKTNKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEEKFN EKGEVSEKIITMADGTRLEYTGIKSDGTGKAKYVLKNFTLEGKVANDKTTLEVKEGTVTLSM NISKSGEVSVELNDTDSSAATKKTAAWNSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAG TNLEGTAVEIKTLDELKNALKLEHHHHHH SEQIDNO:41 ChimericOspASerotype5/Serotype3,N-terminallipidation, His-tagged,includingtheOspBlipidationsignalsequence: MRLLIGFALALALIG(SEQIDNO:14)whichiscleavedduring processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMKVLVSKEKDKNGKYSLMATVEKLELKG TSDKNNGSGTLEGEKTNKSKVKLTIAEDLSKTTFEIFKEDGKTLVSKKVTLKDKSSTEEKFNE KGEISEKTIVMANGTRLEYTDIKSDKTGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVTLSM NISKSGEITVALDDTDSSGNKKSGTWDSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGN ALEGSPAEIKDLAELKAALKLEHHHHHH SEQIDNO:42 ChimericOspASerotype6/Serotype4,N-terminallipidation, His-tagged,includingtheOspBlipidationsignalsequence: MRLLIGFALALALIG(SEQIDNO:14)whichiscleavedduring processing MRLLIGFALALALIGCAQKGAESIGSVSVDLPGGMTVLVSKEKDKNGKYSLEATVDKLELKG TSDKNNGSGTLEGEKTNKSKVKLTIADDLSQTKFEIFKEDAKTLVSKKVTLKDKSSTEEKFNE KGETSEKTIVMANGTRLEYTDIKSDGSGKAKYVLKDFTLEGTLAADGKTTLKVTEGTVVLSM NILKSGEITVALDDSDTTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGT NLEGNAVEIKTLDELKNALKLEHHHHHH SEQIDNO:43 SID1 FNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLS KNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSN GTKLEGSAVEITKLDEICNALK SEQIDNO:44 S2D1 FNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVT LSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYD SAGTNLEGTAVEIKTLDELCNALK SEQIDNO:45 S3D1 FNDKGKLSEKVVTRANGTRLEYTEIKNDGSGKAKEVLKGFALEGTLTDGGETKLTVTCGTVT LSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNR AGNALEGSPAEIKDLAELCAALK SEQIDNO:46 S4D1 FNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLS KHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAG TNLEGNAVEIKTLDELCNALK SEQIDNO:47 S5D1 FNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTL SKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAG TNLEGKAVEITTLKELCNALK SEQIDNO:48 S6D1 FNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVV LSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDS AGTNLEGKAVEITTLKELCNALK SEQIDNO:49 S3HYBD1(BVA) FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTV TLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYN RAGNALEGSPAEIKDLAELCAALK SEQIDNO:50 BVAD1 FNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTTLKITCGTVT LSKHIAKSGEVTVEINDTSSTPNTKKTGKWDARNSTLTIIVDSKNKTKLVFTKQDTITVQSYNP AGNKLEGTAVEIKTLQELCNALK SEQIDNO:51 S3hybD1(Bsp):hybridOspAC-terminalfragment;aminoacids 126-175fromBorreliaspielmaniiandaminoacids177-274 fromBorreliagarinii,strainPBr,withdisulfidebondtype 1andTinposition233 FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKTKLTVTCGTVTLS KNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRA GNALEGSPAEIKDLAELCAALK SEQIDNO:52 MSPD1 FNEKGELSEKTLVRANGTKLEYTEIKSDGTGKAKEVLKDFTLEGTLANEKATLTVKCGTVTL SKNIDKSGEVTVALNDTDSTAATKKTGAWDSKTSTLTITVNSKKTKDLVFTKQDTITVQKYD SAGTTLEGSAVEIKTLDELCNALK SEQIDNO:53 Forwardprimerforthe16S-23Sintergenicspacer GTATGTTTAGTGAGGGGGGTG SEQIDNO:54 Reverseprimerforthe16S-23Sintergenicspacer GGATCATAGCTCAGGTGGTTAG SEQIDNO:55 Forwardnestedprimerforthe16S-23Sintergenicspacer AGGGGGGTGAAGTCGTAACAAG SEQIDNO:56 Reversenestedprimerforthe16S-23Sintergenicspacer GTCTGATAAACCTGAGGTCGGA SEQIDNO:57 ForwardprimerfortheRecAgeneofBorrelia CATGCTCTTGATCCTGTTTA SEQIDNO:58 ReverseprimerfortheRecAgeneofBorrelia CCCATTTCTCCATCTATCTC SEQIDNO:59 25-merpeptidefromtheInvariableRegion6(IR6)ofVlsE MKKDDQIAAAMVLRGMAKDGQFALK SEQIDNO:60 Mousecathelin RLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE SEQIDNO:61 KLKpeptide KLKLLLLLKLK SEQIDNO:62 N-terminalpeptideforlipidation CKQN SEQIDNO:63 5-(dIdC).sub.13-3 dIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdCdIdC

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