PROTEINS USED FOR THE DIAGNOSIS OF LYME BORRELIOSIS

Abstract

Chimera proteins includlng: (i) at least one sequence of a DbpA protein of a Borrelia species selected from B. afzelii, B. burgdorferi sensu stricto and B. garinii, and (ii) at least one sequence of an OspC protein of a Borrelia species selected from B. afzelii, B. burgdorferi sensu stricto and B. garinii. Also, a method and a kit for the in vitro diagnosis of Lyme borreliosis using said proteins.

Claims

1. A polypeptide comprising (i) a Decorin-binding protein A (DbpA) extracellular domain portion from B. afzelii, B. burgdorferi sensu stricto, or B. garinii that includes DbpA immunodominant epitope regions, and (ii) an Outer-surface protein C (OspC) extracellular domain portion from B. afzelii, B. burgdorferi sensu stricto, or B. garinii that includes OspC immunodominant epitope regions.

2. The polypeptide as claimed in. claim 1, wherein the DbpA extracellular domain portion is on the N-terminal side of the polypeptide, and the OspC extracellular domain portion is on the C-terminal side of the polypeptide.

3. The polypeptide as claimed in claim 1, further comprising a linking region between the DbpA extracellular domain portion and the OspC extracellular domain portion.

4. A polypeptide comprising (i) a first amino acid sequence having at least 40% sequence identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 6, and (ii) a second amino acid sequence having at least 50% sequence identity with any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 4, and 7, wherein the first amino acid sequence and the second amino acid sequence are capable of forming an immunological complex with antibodies produced following a Borrelia infection.

5. The polypeptide as claimed in claim 4, wherein the first amino acid sequence is on the N-terminal side of the polypeptide, and the second amino acid sequence is on the C-terminal side of the polypeptide.

6. The polypeptide as claimed in. claim 4, further comprising a linking region between the first amino acid sequence and the second amino acid sequence.

7. The polypeptide as claimed in claim 4, further comprising a third amino acid sequence of at least 6 contiguous histidines at the N-terminal or C-terminal end.

8. A nucleic acid encoding the polypeptide as claimed in claim 4.

9. A polypeptide comprising (i) a first amino acid sequence having at least 40% sequence identity with the amino acid sequence of SEQ ID NO: 1, and (ii) a second amino acid sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein the first amino acid sequence and the second amino acid sequence are capable of forming an immunological complex with antibodies produced following a Borrelia infection.

10. A diagnostic kit comprising the polypeptide as claimed in claim 1.

11. The diagnostic kit as claimed in claim 10, further comprising an anti-human-immunoglobulin labeled with a label.

12. The diagnostic kit as claimed in claim 10, further comprising a chimeric Variable Lipoprotein Surface-Exposed (VlsE) polypeptide.

13. A diagnostic kit comprising the polypeptide as claimed in claim 4.

14. The diagnostic kit as claimed in claim 13, further comprising an anti-human-immunoglobulin labeled with a label.

15. The diagnostic kit as claimed in claim 13, further comprising a chimeric Variable Lipoprotein Surface-Exposed (VlsE) polypeptide.

16. A. method for the in vitro diagnosis of Lyme borreliosis using a biological sample, comprising: bringing the biological sample into contact with at least one polypeptide as claimed in claim 1; and determining whether there is formation of an immunological complex between the polypeptide and antibodies of the biological sample.

17. The method as claimed in claim 16, wherein formation of the immunological complex is determined by adding at least one anti-human-immunoglobulin labeled with a label.

18. The method as claimed in claim 16, wherein the polypeptide is immobilized on a solid support.

19. A method for the in vitro diagnosis of Lyme borreliosis using a biological sample, comprising: bringing the biological sample into contact with at least one polypeptide as claimed in claim 4; and determining whether there is formation of an immunological complex between the polypeptide and antibodies of the biological sample.

20. The method as claimed in claim 19, wherein formation of the immunological complex is determined by addina at least one anti-human-immunoglobulin labeled with a label.

Description

EXAMPLES

Example 1

Preparation of the Plasmid Constructs Encoding the DpbA-OspC Chimeric Recombinant Proteins

[0054] The DNA sequences encoding the various DpbA and OspC sequences described are identified in table 1. The DNA sequences were optimized in order to promote expression in E. coli using Geneoptimizer? and synthesized respectively by GenScript corporation (Scotch Plains, N.J., USA) or GeneArt GmbH (Regensburg, Germany).

TABLE-US-00001 TABLE 1 Sequence origin B. burgdorferi species *Isolate; **amino acids (aa); ***GenBank accession No. protein B. sensu stricto B. afzelii B. garinii DbpA *B31; **aa 2-192; *PKo; **aa 2-150; *40; **aa 2-187; ***AF069269 ***AJ7131967 ***AF441832 *PBi; **aa 2-176; ***AJ841673 OspC *B31; **aa 26-210; *PKo; **aa 2-212; *PEi; **aa 32-208; ***X73622 ***X62162 ***AJ749866

[0055] Each chimeric recombinant protein comprises at least one epitope region corresponding to the extracellular domain of a DbpA sequence of Borrelia burgdorferi sensu stricto or B. afzelii or B. garinii and at least one epitope region corresponding to the extracellular domain of an OspC sequence of Borrelia burgdorferi sensu stricto or B. afzelii or B. garinii.

[0056] The combinations of various nucleotide sequences encoding DbpA and/or OspC sequences and also the modifications of nucleotide sequences, such as deletions, addition of a linking sequence or addition of a linker sequence, were carried out by genetic engineering using the PCR techniques well known to those skilled in the art and described, for example, in Sambrook J. et al., Molecular Cloning: A. Laboratory Manual, 1989.

[0057] The DNA sequences encoding the chimeric proteins of interest were introduced into the pMR expression vector [2] between the BamHI restriction site in the 5 position and the EcoRI or HindIII site in the 3 position. The plasmid constructs and the corresponding proteins cited as example (bLYM114, bLYM120 and bLYM121) are described in table 2. The presence of MRGS in the N-terminal position of the recombinant proteins and the corresponding nucleotide sequence ATG AGG GGA TCC was introduced by the cloning technique used into the pMR expression vector. Only the ATG start codon and consequently the Met amino acid are really essential in this sequence.

[0058] A poly-histidine sequence (6?His) was introduced on the N-terminal side of each recombinant protein. This sequence allows purification of the recombinant proteins on a metal-chelate affinity column. It is a region for attachment to the Ni-NTA gel which makes it possible to subsequently facilitate the step of purifying the chimeric recombinant protein. This HHHHHH peptide (SEQ ID NO: 22) is encoded by the nucleotide sequences CATCATCATCATCATCAT (SEQ ID NO: 23) or CATCATCATCATCATCAC (SEQ ID NO: 24) or CATCATCACCACCATCAT (SEQ ID NO: 25) or by any other sequence encoding the sequence SEQ ID NO: 22. This particular attachment region, comprising a succession of histidines, allows in particular the oriented attachment of the recombinant protein to a support consisting of silica or of metal oxides.

TABLE-US-00002 TABLE 2 Plasmid constructs and corresponding proteins Plasmid construct characteristics Recombinant protein characteristics Site of insertion of the N-terminal Parental insert sequence into the Name Tag B. burgdorferi sequence vector vector bLYM114 6 x His B. afzelii strain PKo pMR78* 5BamHI/3EcoRI SEQ ID DbpA aa 2-150 + NO: 9 OspC aa 2-212 bLYM120 6 x His B. sensu stricto strain B31 pMR78* 5BamHI/3HindIII SEQ ID DbpA as 28-192 + NO: 11 OspC aa 26-210 bLYM121 6 x His B. garinii pMR78* 5BemHI/3HindIII SEQ ID DbpA III aa 25-187 strain 40 + NO: 14 DbpA IV aa 24-176 strain PBi + OspC as 32-208 strain PEi *[2]

Example 2

Expression of the Recombinant Proteins bLYM114, bLYM120 and bLYM121 of Example 1 and Purification

[0059] A plasmid construct corresponding to a sequence SEQ ID NO: 16, 18 or 21 inserted into an expression vector (pMR) was used to transform an E. coli bacterium (strain BL21) according to a conventional protocol known to those skilled in the art. The transformed bacteria were selected by virtue of their ampicillin resistance carried by the pMR vector.

[0060] A clone of a recombinant bacterium was then selected in order to inoculate a preculture of 40 ml of 2?YT medium (16 g/l tryptone; 10 g/l yeast extract; 5 g/l NaCl, pH 7.0) containing 100 ?g/ml of ampicillin. After 15 to 18 hours of incubation at 30?C. with shaking at 250 rpm, this preculture was used to inoculate 1 liter of 2?YT medium containing 2% glucose and 100 ?g/ml of ampicillin. This culture was incubated at 30? C. with shaking at 250 rpm until the OD at 600 nm reaches 1.0/1.2. The culture was maintained for 3 hours 30 min. or 4 hours at 30? C. while adding 0.4 mM isopropyl-?-D-thiogalactopyranoside (IPTG), and harvested by centrifugation at 6000 g for 30 min. The cell pellet was stored at ?60? C. For the purification, the wet biomass was thawed and resuspended in a lysis buffer containing protease inhibitors without EDTA (Roche) and benzonase nuclease (Novagen), and subjected to cell rupture at. 1.6 kBar in a cell disruptor (Constant Systems Ltd, Daventry, United Kingdom). The lysate was then centrifuged at 10 000 rpm for 45 min. at 2-8? C. The supernatant obtained contains the soluble proteins. This supernatant was filtered through a 0.45? filter and purified by affinity chromatography on a metal chelation column (nickel-nitrilotriacetio acid matrix (Ni-NTA, Qiagen)). To do this, the supernatant was loaded (1 ml/min) at 18-25? C. onto an 8 ml column of Ni-NTA gel equilibrated in buffer A (see table 3). The column was then washed in buffer A, until an OD.sub.230 nm=0 was obtained at the column outlet. The elution of the recombinant protein is obtained by applying a buffer B, according to the indications reported in. table 3, and the purified protein was dialyzed in a 10000 ou 20000 MWCO dialysis cassette (Slide-A-Lyser?, Pierce) against a dialysis buffer. The conditions for purification on Ni-NTA gel are described in table 3.

TABLE-US-00003 TABLE 3 Recombinant protein purification bLYM121 bLYM114 bLYM120 SEQ ID Protein SEQ ID NO: 14 SEQ ID NO: 11 NO: 14 Lysis and Buffer A.sup.1 washing buffer Elution buffer Buffer B.sup.2 Elution step 1 90% buffer A + 92% buffer A + 100% buffer B 10% buffer B 8% buffer B (4CV) (4CV) Elution step 2 100% buffer B 100% buffer B NA Purification 12 13 20 yield mg protein/g wet biomass Purification 80 122 245 yield mg protein/L of culture .sup.150 mM sodium phosphate, 30 mM imidazole, 500 mM NaCl, 0.1% TWEEN 20, 5% glycerol, pH = 7.8 .sup.250 mM sodium phosphate, 325 mM imidazole, 500 mM NaCl, 5% glycerol, pH = 7.5

[0061] The samples were analyzed on. NuPAGE? Novex? 4-12% in a NuPAGE? MES-SDS buffer, according to the instructions of the producer (Invitrogen). The proteins were either stained. with COOMASSIE BRILLIANT BLUE or were transferred electro-phoretically onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) dry milk in PBS and incubated with an antipentahistidine antibody (Qiagen) in PBS containing 0.05% TWEEN 20. A horseradish peroxidase-labeled goat anti-mouse IgG conjugate (Jackson Immunoresearch laboratories) in PBS/TWEEN was used as secondary antibody.

[0062] The protein concentration was determined using the Bradford kit (PIERCE COOMASSIE PLUS, Perbio Science) with BSA as protein standard.

Example 3

Detection of Human IgGs and IgMs With the Chimeric Recombinant Proteins Using a Line Immunoblot Technique

[0063] Each recombinant protein was deposited on a polyvinylidene difluoride membrane (PVDF, Immobilon, Millipore, Bedford, Mass. USA) according to the following protocol:

[0064] The protein concentration was adjusted to 1 mg/mi in PBS, pH 7.2, and diluted in PBS, pH 7.2, supplemented with 0.03% TWEEN 20 (dilution 1/200.sup.th). The PVDF membrane was wetted in methanol, washed in demineralized water and laid out on a wet blotting paper. A plastic ruler was immersed in the protein dilution and attached to the PVDF membrane. After depositing of the proteins and drying of the membranes, the membranes were cut vertically into narrow strips. Before use, the narrow strips were incubated with 5% gelatin in TBS, pH 7.5, for 1 hour at 37? C. The immunoblot protocols were carried out at ambient temperature as described by Bretz A. G. et al. [3]. The narrow strips were incubated for 2 hours with human sera diluted to 1/200.sup.th in TES with. 1% gelatin, washed and incubated with an anti-human-IgG or anti-human-IgM antibody labeled with alkaline phosphatase (Sigma, St-Louis, USA) diluted to 1/1000.sup.th in TES with 1% gelatin. After washing, the narrow strips were incubated with the alkaline phosphatase substrate BCIP-NET (KPL, Gaithersburg, Md., USA) for 30 min., and then washed in distilled water and dried.

Panel of Sera Tested

[0065] The human sera were collected from clinically well-defined, typical LB patients corresponding to the various stages of LB (22 with erythema migrans [EM], 5 with carditis, 20 with neuroborreliosis [NE], 20 with Lyme arthritis [LA], 20 with acrodermatitis chronica atrophicans [ACA] and 10 with lymphadenosis cutis benigna [LCE]), Anti--Lyme IgGs were found by immunoblot described previously and using whole cell lysates [4], in the sera of patients with LA, ACA and carditis. EM, NB and LCB were identified clinically, but not all the corresponding sera were found to be positive by in-house immunoblot [4], or using the commercially available kits (Vidas? Lyme (biom?rieux) Borrelia IgG (Diasorin?) and Borrelia igM (r-biopharm?)). On the other hand, all the cases of NB included in the study had detectable antibodies in the cerebrospinal fluid [CSF] (index extending from 2 to 27.1 with Vidas? Lyme (biom?rieux)) The presence of IgM was sought only in the stage I and stage II clinical cases and not in the chronic stages.

[0066] The negative control group consisted of 31 sera previously found to be negative for the presence of anti-Lyme antibodies in conventional assays. Furthermore, 64 sera from healthy blood donors residing in a region endemic for Lyme disease (Monthley, Valais, Switzerland) were tested with the recombinant protein.

[0067] The strength of the reaction was evaluated as follows: [+], [++], [+++], [?] or equivocal results. The equivocal results were considered to be negative.

[0068] The results are given in table 4.

TABLE-US-00004 TABLE 4 Reactivity in Line immunoblot of human sera from patients with Lyme borreliosis, with 3 chimeric recombinant proteins bLYM114 (SEQ ID NO: 9), bLYM120 (SEQ ID NO: 11) and bLYM121 (SEQ ID NO: 14) IgG IgM Stage I Stage II Stage III Stage I Stage II EM NB Carditis LA ACA LCB EM NB Carditis Protein (n = 22) (n = 20) (n = 5) (n = 19) (n = 20) (n = 10) (n = 22) (n = 20) (n = 5) bLYM114 5 10 0 7 12 2 7 7 2 bLYM120 6 7 0 8 6 0 11 7 2 bLYM121 2 10 5 9 8 0 7 7 2 ? bLYM 9 13 5 18 17 2 11 7 2 114 + 120 + 121 Positive 40.9% 59.1% 100% 94.7% 85% 20% 50% 35% 40% sera (%) 1[+++] 8[+++] 4[+++] 7[+++] 8[+++] 1[++] 1[+++] 5[++] 2[++] arid 4[++] 2[++] 1[+] 8[++] 5[++] 1[+] 7[++] 2[+] reaction 4[+] 3[+] 3[+] 4[+] 5[+] strength Total 66.7% 42.5% positives 28[+++] 1[+++] and 20[++] 14[++] reaction 16[+] 7[+] strength

[0069] The specificity is 100% on the basis of 31 sera originating from healthy individuals determined to be Lyme-negative using the standard commercially available tests.

[0070] IgGs Detection

[0071] The results indicate that the recombinant chimeric fusion proteins are diagnostic tools that are sensitive at all stages of the infection. for IgGs and IgMs. They demonstrate an additional effect of the three recombinant proteins based, respectively, on sequences of Borrelia afzelii, B. sensu stricto and B. garinii for the detection of IgGs. The combined use of the three chimeric recombinant proteins makes it possible, at stage I of the infection, to detect IgGs in 9 cases of patients with EM out of 22 (i.e. 40.9% sensitivity).

[0072] IgM Detection

[0073] Anti-chimera protein IqMs are found in 11 cases out of 22 (i.e. 50% sensitivity). These chimera proteins therefore detect the IgMs more often than the IgGs in the sera of stage-I LB patients. The tests performed as a control: in-house immunoblot [4], and the commercially available kit Borrelia IgM (r-biopharm?) do not further detect IgM-positive sera. In addition, 3 sera found to be neaative using the immunoblot test and Borrelia IgM (r-biopharm?) are detected by the three chimeric proteins cited as example (3/3) or by one of the three proteins cited as example (1/3). The combined use of the three recombinant proteins makes it possible to improve the IgM detection sensitivity by 13.6% in stage I of the infection

Example 4

Preparation of Plasmid Constructs Encoding the VlsE Chimeric Recombinant Proteins

[0074] The DNA sequences encoding the various sequences of the protein are identified in table 5.

TABLE-US-00005 TABLE 5 Sequence origin B. burgdorferi species *Isolate; **amino acids (aa); ***GenBank accession No. protein B. sensu stricto B. afzelii B. garinii V1sE *PBi; **aa 20-293; ***AJ630106 (GenScript Corp) IR6 *B31; **aa 274-305; *ACA-1; **aa 172-188; *Ip90; **aa 167-191; ***U76405 ***U76405 ***AAN87834 (GeneArt GmbH) (GeneArt GmbH) (GeneArt GmbH)

[0075] The sequences were optimized for their expression in E. coli using GeneOptimizer? and synthesized respectively by GenScript corporation (Scotch Plains, N.J., USA) or GeneArt GmbH (Regensburg, Germany).

[0076] Additional modifications to the DNA, deletions or combinations of various sequences were carried out by PCR by genetic engineering using the PCR techniques well known to those skilled in the art and described, for example, in Sambrook J. et al., Molecular Cloning: A Laboratory Manual, 1989. The DNA sequences were ligated into the pMR [2] or pET-3d (Novagen?) expression vector. The plasmid constructs and the corresponding proteins cited as example (bLYM110, bLYM125) are described in table 6.

TABLE-US-00006 TABLE 6 Plasmid constructs and corresponding proteins Plasmid construct characteristics Recombinant protein characteristics Site of insertion of the N-terminal Parental insert sequence into the Name Tag B. burgdorferi sequence vector vector bLYM110 6 x His V1sE garinii pBi as 20-293 + pMR78 5BamHI/3HindIII SEQ ID 3 IR6 [sensu stricto B21 as NO: 39 274-305 + afzelii ACA-laa bLYM125 8 x His 172-188 + pET-3d 5NcoI/3BamHI SEQ ID garinii Ip90 aa 167-191] NO: 41

Example 5

Expression of the Recombinant Proteins of Example 4 and Purification

[0077] A plasmid construct described in example 4 was used to transform an E. coli bacterium (strain BL21) according to a conventional protocol known to those skilled in the art. The transformed bacteria were selected by virtue of their ampicillin resistance carried by the pMR or pET vector.

[0078] A clone of a recombinant bacterium was then selected in order to inoculate a preculture of 40 ml of 2?YT medium (16 g/l tryptone; 10 g/l yeast extract; 5 g/l NaCl, pH 7.0) containing 100 ?g/ml ampicillin. After 15 to 18 hours of incubation at 30? C. with shaking at 250 rpm, this preculture was used to inoculate 1 liter of 2?YT medium containing 2% glucose and 100 ?g/ml ampicillin. This culture was incubated at 30? C. with shaking at 250 rpm until the OD at 600 nm reaches 1.0/1.2. The culture was maintained for 3 hours 30 min. or 4 hours at 30? C. while adding 0.4 mM isopropyl-?-D-thio-galactopyranoside (IPTG) and harvested by centrifugation at 6000 g for 30 min. The cell pellet was stored at ?60? C. For the purification, the wet biomass was resuspended in a lysis buffer containing protease inhibitors without EDTA (Roche) and benzonase nuclease (NovagenS), and subjected to cell rupture at 1.6 kBar in a cell disrupter (Constant Systems Ltd, Daventry, United Kingdom). The lysate was then centrifuged at 10 000 rpm for 45 minutes at 2-8? C. After filtration through a 0.22 ?m filter, the supernatant was loaded onto an Ni-NTA column (Qiagen?) equilibrated in a lysis buffer. The resin was then washed with the same buffer until the A.sub.280 nm reached the base line. An elution was carried out with the elution buffer, and the purified protein was dialyzed in a Pierce Slide-A-Lyser? 10000 or 20000 MWCO dialysis cassette against the dialysis buffer. The conditions for purification on Ni-NTA gel are described in table 7.

TABLE-US-00007 TABLE 7 Recombinant protein purification bLYM110 bLYM125 Protein SEQ ID NO: 39 SEQ ID NO: 41 Lysis and washing buffer Buffer A.sup.1 Buffer A.sup.1 + 2M urea Elution buffer Buffer B.sup.2 Buffer B.sup.2 modified with 600 mM imidazole Elution step 1 86% Buffer A + 14% Buffer B 92% Buffer A + 8% Buffer B (4CV) (4CV) Elution step 2 100% Buffer B 100% Buffer B Purification yield 0.5 0.8 mg protein/g wet biomass Purification yield 8.7 17 mg protein/L of culture .sup.150 mM sodium phosphate, 30 mM imidazole, 500 mM NaCl, 0.1% TWEEN 20, 5% glycerol, pH = 7.8 .sup.250 mM sodium phosphate, 325 mM imidazole, 500 mM NaCl, 5% glycerol, pH = 7.5

[0079] The samples were analyzed on NuPAGE1 Novex? 4-12% in a NuPAGE? NES-SDS circulating buffer, according to the instructions of the producer (Invitrogen?). The proteins were either stained with COOMASSIE BRILLIANT BLUE or were transferred electrophoretically onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) dry milk in PBS and incubated with an anti-pentahistidine antibody (Qiagene) in PBS containing 0.05% TWEEN 20. A horseradish peroxidase-labeled goat anti-mouse IgG conjugate (Jackson immunoresearch laboratories) in PBS/TWEEN was used as secondary antibody.

[0080] The protein concentration was determined using the Bradford Assay Kit (PIERCE COOMASSIE PLUS, Perbio Science) with BSA as protein standard.

Example 6

Detection of Human IgGs and IgM With the Chimeric Recombinant Protein bLYM110 of Example 5 Using a Line Immunoblot Technique

[0081] The recombinant protein was deposited onto a polyvinylidene difluoride membrane (PVDF, Immobilon, Millipore?, Bedford, Mass. USA) according to the following protocol: [0082] The protein concentration was adjusted to 1 mg/mI in PBS, pH 7.2, and diluted in PBS, pH 7.2, supplemented with 0.03% TWEEN 20 (dilution 1/200.sup.th). The PVDF membrane was wetted in methanol, washed in demineralized water and laid out on a wet blotting paper. A plastic ruler was immersed in the protein dilution and attached to the PVDF membrane. After depositing of the proteins and drying of the membranes, the membranes were cut vertically into narrow strips. Before use, the narrow strips were incubated with 5% gelatin in TBS, pH 7.5, for 1 hour at 37? C. The immunoblot protocols were carried out at ambient temperature as described by Bretz A. G. et al. [3]. The narrow strips were incubated for 2 hours with human sera diluted to 1/200.sup.th in TBS with 1% gelatin, washed and incubated with anti-human IgGs or IgMs labeled with alkaline phosphatase (Sigma?, St-Louis, USA) diluted to 1/1000.sup.th in TBS with 1% gelatin. After washing, the narrow strips were incubated with the BCIP-NBT substrate (KPL, Gaithersburg, Md., USA) for 30 minutes, washed in distilled water and dried.

Panel of Sera Tested

[0083] The human sera were collected from clinically well-defined, typical LB patients corresponding to the various stages of LB (22 with erythema migrans [EM], 5 with carditis, 20 with neuroborreliosis [NB], 20 with Lyme arthritis [LA], 20 with acrodermatitis chronica atrophicans [ACA] and 10 with lymphadenosis cutis benigna [LCB]). Anti-Lyme IgGs were found by immunoblot, described previously using whole cell. lysates [4], in the sera of patients with LA, ACA and carditis. EM, NB and LCB were identified clinically, but not all the corresponding sera were found to be positive using the immunoblot [4], or using the commercially available kits (Vidas? Lyme (biom?rieux?), Borrelia TqG (Diasorin?) and Borrelia IgM (r-biopharm?)). On the other hand, all the cases of NB included in the study had detectable antibodies in the cerebrospinal fluid [CSF] (index extending from 2 to 27.1).

[0084] The negative control group consisted of 31 sera previously found to be negative for the presence of anti-Lyme antibodies in conventional assays. Furthermore, 64 sera from healthy blood donors residing in a region endemic for Lyme disease (Monthley, Valis, Switzerland) were tested with the recombinant protein. The strength of the reaction was evaluated as follows: [+], [++], [+++], [?] or equivocal results. The equivocal results were considered to be negative.

[0085] The results are given in table 8 below.

TABLE-US-00008 TABLE 8 Stage I Stage II Stage III EM NB Carditis LA ACA Lymph. Donors (n = 22) (n = 20) (n = 5) (n = 19) (n = 20) (n = 10) (n = 64) IgG 17 20 5 19 20 9 6 77.3% 100% 100% 100% 100% 90% 9.4% 12[+++] 11[+++] 4[+++] 13[+++] 20[+++] 3[+++] 6[+] 4[++] 7[++] 1[++] 4[++] 2[++] 1[+] 2[+] 2[+] 4[+] Total IgG positives 93.7% IgM 5 4 2 1 22% 20% 40% 1.5% 1[++] 2[++] 1[++] 1[+] 4[+] 1[+] 1[+] Total IgM positives 23.4%

[0086] IgG Detection

[0087] The results indicate that the recombinant protein bLYM110 is a diagnostic antigen that is highly sensitive at all stages of the infection for IgGs. At stage I of the infection, the IgGs were detected in 17 cases of patients with EM out of 22 (i.e. 77.3% sensitivity). Five of the patients with EM who are found to be negative with the recombinant protein are also found to be negative with the in-house immunoblot and with the commercially available kits. Seven EM sera found to be positive with the recombinant protein were not detected by immunoblot, which represents a 31.8% improvement in sensitivity with the recombinant protein. At the primary stage of the infection, in the absence of characteristic redness, the diagnosis can be difficult since the other clinical manifestations of Lyme disease are not specific. Furthermore, only a few patients with EM are detected using the conventional tests. Therefore, the protein of the invention improves the detection of IgGs at stage I of the infection, bringing their detection to more than 77% in patients with EM.

[0088] IgM Detection

[0089] Anti-chimera protein IgMs are found in 23.4% of the LB sera. The protein detects the IgGs more often than the IgMs in the sera of stage-I and -II LB patients.

Example 7

Evaluation and Validation of the Chimeric Recombinant Proteins bLYM114, bLYM120, bLYM121 and bLYM125 in a VIDAS? test (bioM?rieux)

[0090] This validation is carried out in a VIDASO test using: [0091] 1) the chimeric recombinant proteins bLYM114, bLYM120 and bLYM121, obtained according to examples 1 and 2, for the IgM detection, and [0092] 2) the chimeric recombinant proteins bLYM114 and bLYM120, obtained according to examples 1 and 2, and the chimeric protein bLYM125, obtained according to examples 4 and 5, for the IgG detection.

[0093] The principle of the VIDAS? test is the following: a tip constitutes the solid support which also serves as a pipetting system for the reagents present in the strip. The recombinant protein(s) is (are) attached to the tip. After a dilution step, the sample is drawn up and forced back several times in the tip. This allows the anti-Lyme immunoglobulins in the sample to bind to the recombinant proteins. The unbound proteins are removed by washing. An anti-human-immunoglobulin antibody conjugated to alkaline phosphatase (ALP) is incubated in the tip, where it binds to the anti-Lyme immunoglobulins. Washing steps remove the unbound conjugate. During the final visualizing step, the alkaline phosphatase (ALP) substrate, 4-methylumbelliferyl phosphate, is hydrolyzed to 4-methyl-umbelliferone, the fluorescence of which emitted at 450 nm is measured. The intensity of the fluorescence is measured by means of the Vidas? optical system and is proportional to the presence of anti-Lyme immunoglobulins present in the sample.

[0094] The results are analyzed automatically by the VIDAS? and expressed as RFV (Relative Fluorescent Value).

[0095] 255 positive sera (equivocal sera positive sera) and 298 negative sera (equivocal+negative) were thus assayed with the Vidas? system.

[0096] The Vidas? Lyme IgG tips are sensitized with 300 ?L of solution comprising the bLYM114, bLYM120 and bLYM125 proteins of the invention, each. at a concentration of 1 ?g/mL in a common sensitizing solution.

[0097] In the first step, the sera are incubated for 5.3 min. for the formation of the antigen-antibody complexes. In the second step, anti-human-IgGs labeled with ALP are incubated for 5.3 min.

[0098] The results are given as an index relative to a positivity threshold positioned at 135 RFV in the protocol. [0099] Among the 255 positive sera tested, 246 are positive and 9 are falsely negative, which corresponds to a sensitivity of 96.5%. [0100] Among the 298 negative sera tested, 284 are negative and 14 are falsely positive, which corresponds to a specificity of 95.3%.

LITERATURE REFERENCES

[0101] 1. G?ttner G. et al., Int. J. Microbiol. 293, Suppl. 172-173 (2004) [0102] 2. Arnaud N. et al., Gene 1997; 199:149-156. [0103] 3. Bretz A. G., K. Ryffel, P. Butter, E. Dayer and O. P?ter. Specificities and sensitivities of four monoclonal antibodies for typing of Borrelia burgdorferi sensu lato isolates. Clin. Diag. Lab. Immunol. 2001; 8: 376-384. [0104] 4. Ryffel K., P?ter O., Rutti B. and E. Dayer. Scored antibody reactivity by immunoblot suggests organotropism of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii and B. valaisiana in human. J. Clin. Microbiol. 1999; 37:4086-92