Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to vaccines against Lyme disease, in particular vaccines comprising one or more polypeptides isolated from Borrelia bu rdorferi ss, Borrelia afzelii or Borrelia garinii.
• Lyme borreliosis also known as Lyme disease
• Lyme disease is a vector-borne disease transmitted by a hard tick of the genus Ixodes. It occurs mainly in the Northern Hemisphere where it is the most common vector-borne disease. Recent data also suggest that its range also extends into the southern hemisphere with human cases in Australia (Mayne et al., 201 1 [1]) and ticks infected with Borrelia identified in South America ( Barbieri et al., 2013 [2]).
• the bacterium responsible for borreliosis is a spirochete belonging to the group Borrelia burgdorferi sensu lato with about 20 identified species.
• Lyme borreliosis usually develops in wildlife in a wide range of vertebrate hosts and is manifested accidentally in humans first by skin inflammation, erythema migrans, then by a wide variety of clinical manifestations: articular, cardiac, neurological and cutaneous (Radolf et al., 2012
• the present invention precisely meets the aforementioned needs of the prior art, by providing vaccine compositions for the prevention of Lyme disease.
• the inventors have developed a proteomic approach to identify and select effective polypeptides for the prevention of Lyme disease. This approach was carried out on the basis of three species of Borrelia that the inventors have determined to be the most involved in human and animal pathology, in particular in dogs, namely Borrelia burgdorferi ss, Borrelia afzelii and
• the subject of the present invention is in particular a vaccine composition
• a vaccine composition comprising at least one Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide chosen from the sequences described below.
• sequences SEQ ID NO: 1 to 92 shown in Table 1 below in which the sequence numbers of the appended sequence listing, the names of the corresponding polypeptides and the names of the corresponding loci in the genome of the invention are described.
• Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii are described.
• Table 1 describes the isolated polypeptides consisting of a sequence chosen from SEQ ID NO: 1 to 92 that may be used in the vaccine composition of the invention.
• Borrelia burgdorferi means “Borrelia burgdorferi ss” or “Borrelia burgdorferi sensu stricto” as opposed to “Borrelia burgdorferi sensu lato” which covers about 20 different species.
• sequence-determining letters of the polypeptides described herein correspond to the one-letter abbreviation proposed by Leder (Leder et al., Introduction to Molecular Medicine, Ed Scientific American, 1994 [9]).
• the subject of the present invention is in particular a vaccine composition
• a vaccine composition comprising at least one Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide chosen from the sequences SEQ ID NO: 1 to 92.
• the at least one polypeptide is chosen. among the sequences SEQ ID NO: 10 to 92, preferably 10 to 18, preferably 10 to 15.
• Any polypeptide of sequence SEQ ID NO: 1 to 92, or any combination of at least two of the polypeptides of sequence SEQ ID NO: 1 to 92, may be used in the vaccine composition according to the invention.
• the combination of polypeptides may comprise 2 different polypeptides of sequence SEQ ID NO: 1 to 92, for example 3, 4, 5, 6, 7, 8, 9 or even more than 9 different polypeptides of sequence SEQ ID NO: 1 at 92.
• a combination of the different polypeptides of sequence SEQ ID NO: 1 to 92 may in fact make it possible to increase the therapeutic and / or prophylactic effect of the vaccine composition according to the invention.
• the vaccine composition according to the invention may comprise a combination of two polypeptides as shown in Table 2 below. Table 2: Combination of polypeptide in the vaccine composition according to the invention
• the vaccine composition according to the invention may comprise, in addition to any of the combinations of two polypeptides presented in Table 2 above, at least one third polypeptide different from those of the combination, or a fourth different polypeptide, etc. .
• the vaccine composition comprises a combination of 2, 3, 4, 5, 6 or 7 different polypeptides.
• polypeptides that can be used in the vaccine composition according to the invention are not limited to the polypeptides consisting of the sequence SEQ ID NO: 1 to 92.
• sequences having a homology or an identity with these sequences can also be used. to be used, equivalently, in the vaccine composition according to the invention, since they have the same effect as the polypeptides of sequence SEQ ID NO: 1 to 92, namely an immunogenic effect, useful in the prevention of Lyme disease.
• Those skilled in the art are able to identify homologous sequences from the sequences of the polypeptides that can be used in the vaccine composition according to the invention.
• a sequence used may have a homology or identity greater than 80% with a sequence described in Table 1, for example an identity or homology greater than 85%, or 90%, or 95%, or 99% with a sequence described in Table 1.
• Various methods, well known to those skilled in the art, can be used to determine the homology between several sequences. This may be, for example, the Basic Local Alignment Search Tool (BLAST) method described in Altschul, SF et al., J. Mol. Biol. 1990 [27].
• BLAST Basic Local Alignment Search Tool
• polypeptides on the same line represent the same protein, their amino acid sequences are not strictly identical. This may be due to possible mutations that have occurred distinctly in different species of the genus Borrelia.
• sequences SEQ ID NO: 10, 11 and 12 isolated from the species Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii respectively, represent the same protein.
• Polypeptides that can be used in the vaccine composition according to the invention may be specific to a given Borrelia species, or common to two or three Borrelia species selected from Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii.
• Borrelia burgdorferi ss Borrelia afzelii
• Borrelia garinii For example:
• polypeptides of sequence SEQ ID NO: 1 to 9 are proteins specific to the virulent clone of Borrelia burgdorferi ss;
• polypeptides of sequence SEQ ID NO: 10 to 18 are proteins common to the virulent clones of Borrelia burgdorferi ss,
• polypeptides of sequence SEQ ID NO: 19 to 28 are proteins common to the virulent clones of Borrelia burgdorferi ss and Borrelia garinii;
• polypeptides of sequence SEQ ID NO: 29 to 46 are proteins common to the virulent clones of Borrelia burgdorferi ss and
• polypeptides of sequence SEQ ID NO: 47 to 72 are proteins common to the two virulent clones of Borrelia afzelii and Borrelia garinii;
• polypeptides of sequence SEQ ID NO: 73 to 86 are proteins common to Borrelia burgdorferi ss and Borrelia afzelii;
• polypeptides of sequence SEQ ID NO: 87 to 92 are proteins common to Borrelia afzelii and Borrelia garinii.
• polypeptides of SEQ ID NO: 1 to 9, 13 to 18, 29 to 32, 37, 38, 51, 52, 57, 58, 71, 72 and 87 to 92 are membrane proteins of Borrelia.
• the polypeptides are polypeptides of different sequences.
• the polypeptides represent different proteins.
• the composition comprises a combination of polypeptides of sequence SEQ ID NO: 1 to 92
• the polypeptides may be proteins whose combination makes it possible to obtain immunization simultaneously against two species or the three species Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii.
• the composition according to the invention comprises a protein common to the three aforementioned species or a mixture of several proteins, for example, 2, 3 or 4 proteins, even plus, covering these three species. This embodiment makes it possible to provide universal vaccine compositions with regard to Borrelia populations.
• the vaccine composition according to the invention may comprise at least one polypeptide of Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii chosen from SEQ ID NO: 2, 7 to 15, 19, 20, 25, 26, 29, 30, 33 to 36, 41 to 50, 53, 54, 57 to 72 and 85 to 92.
• the at least one polypeptide is chosen from SEQ ID NO: 10 to 15.
• the vaccine composition may further comprise at least one other Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide selected from SEQ ID NO: 1, 3 to 6, 16 to 18, 21 to 24, 27, 28, 31, 32, 37-40, 51, 52, 55, 56 and 73-84.
• the vaccine composition may further comprise at least one other Borrelia burgdorferi polypeptide, Borrelia afzelii or Borrelia garinii selected from groups (c1), (c2) and (c3),
• said group (d) comprising SEQ ID NO: 19 to 28,
• said group (c2) comprising SEQ ID NOS: 29-46 and 73-86
• said group (c3) comprising SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID NOS: 48-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID NOS: 29-46 and 73-86, said group (c3) comprising SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID
• - is included in one of groups (c1), (c2) or (c3), said at least one other polypeptide is included in a group (c1), (c2) or (c3) different, or
• the vaccine composition comprising at least one Borrelia burgdorferi ss polypeptide, chosen from the sequences SEQ ID NO: 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85.
• the at least one polypeptide is selected from SEQ ID NO: 10.
• the vaccine composition may further comprise at least one other Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide selected from SEQ ID NO: 1, 3 to 7, 1 1, 12, 14 to 18, 20 to 24, 26 to 28, 30 to 32, 34, 36 to 42, 44, 46 to 84 and 86 to 92.
• the vaccine composition may further comprise at least one other Borrelia burgdorferi polypeptide, Borrelia afzelii or Borrelia garinii selected from groups (c1), (c2) and (c3),
• said group (d) comprising SEQ ID NO: 19 to 28,
• said group (c2) comprising SEQ ID NOS: 29-46 and 73-86
• said group (c3) comprising SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID NO : 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85:
• - is SEQ ID NO: 19 and 25, said at least one other polypeptide is included in group (c2) or (c3), or
• polypeptide is SEQ ID NO: 29, 33, 35, 43, 45 or 85, said at least one other polypeptide is included in group (c1) or (c3), or
• composition of the invention may comprise a pharmaceutically acceptable carrier
• pharmaceutically acceptable carrier is understood herein to mean any substance that makes it possible to dilute or transport at least one polypeptide of the vaccine composition according to the invention.
• the pharmaceutically acceptable carrier does not affect the effectiveness of the polypeptide.
• the pharmaceutically acceptable carrier may, for example, be an aqueous solution or an emulsion.
• the pharmaceutically acceptable carrier is an aqueous solution
• it may be, for example, any of the solutions presented in Heitz et al., 2009 (Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Heitz F et al. , Br J Pharmacol 2009 May; 157 (2): 195-206 [10]) or in Wehrle P. (Wehrle P., Galenic Pharmacy, Formulation and Pharmaceutical Technology, 2007 [11]).
• the pharmaceutically acceptable carrier when it is an emulsion, it can be a water-in-oil, oil-in-water, water-in-oil emulsion (Wehrle P. [11]).
• the vaccine composition according to the invention may comprise an adjuvant.
• adjuvant means any substance capable of facilitating and amplifying the immune response to the at least one polypeptide of the vaccine composition according to the invention. It may be for example any adjuvant known to those skilled in the art for the administration of polypeptides.
• the adjuvant may be an adjuvant inducing a humoral response and / or a cellular response.
• the adjuvant may be chosen from alumina hydroxide, saponin extracts, immune stimulation complexes (also called "ISCOM"), inulin, receptor agonists of the type Toll
• TLR Cytosine Phosphate Guanine
• CPG Cytosine Phosphate Guanine
• Chitosan Chitosan or still mycolic acids. It may also be saponin (Roatt et al., 2012 [14]) or alumina hydroxide (Livey et al., 201 1
• the vaccine composition according to the present invention may be used alone or in combination with any known treatment for preventing Lyme disease and / or one or more pathology (s) distinct from Lyme disease.
• the distinct pathology (s) of Lyme disease can be selected from the group consisting of leptospirosis, rabies, distemper, parvovirus and Bordetella infections.
• the term "used in combination” means a use of the vaccine composition according to the invention jointly or concomitantly, concomitantly or sequentially, with any known treatment for preventing Lyme disease.
• the mode of administration may be identical or different depending on the co-administered molecules.
• “Joint or simultaneous” means the use of the composition according to the invention with any known treatment for preventing Lyme disease in a single composition containing them.
• composition means the separate use of the vaccine composition according to the invention and any known treatment for preventing Lyme disease, by the same or different administration routes during the same period of administration.
• “Successive” means the separate use of the vaccine composition according to the invention and any known treatment for preventing Lyme disease, by the same or different administration routes during different periods of administration.
• administration period refers to the time during which a treatment is administered. It may, for example, be several days, for example two days, three days, four days, etc., for example one or more weeks, for example a week, two weeks, three weeks. weeks, etc., for example one or more months, for example one month, two months, three months, etc., for example one or more years, for example one year, two years, three years, etc.
• the present invention also relates to a vaccine composition according to the invention for use as a medicament.
• the present invention also relates to a vaccine composition according to the invention, for use in the prevention of Lyme disease.
• the vaccine composition according to the invention can therefore be used for the manufacture of a medicament, in particular a medicament intended to prevent Lyme disease.
• the vaccine composition for use as a medicament or for use in the prevention of Lyme disease may be for any mammal susceptible to contracting or having contracted Lyme disease. In particular, it may be for humans or dogs, horses, cattle or other ruminants. Preferably, the vaccine composition according to the invention is intended for the prevention of Lyme disease in dogs.
• the vaccine composition according to the invention used as a medicament, may be in any form of appropriate administration. It may be one of the forms known to those skilled in the art to administer an active molecule which is a polypeptide (Peppas NA, Carr DA, Chemical Engineering Science, 64, 4553-4565 (2009) [12]; Morishita M
• the vaccine composition according to the present invention may, for example, be for administration by injection.
• the vaccine composition according to the invention can be packaged in any form known to those skilled in the art in order to be administered by injection. It can be for example a bottle or a bulb.
• the injection may be an intramuscular, intradermal or subcutaneous injection.
• the injection is performed intradermally.
• the injection is carried out intramuscularly or subcutaneously.
• the vaccine composition of the present invention may be administered as a medicament, preferably in an amount sufficient to prevent Lyme disease, particularly to prevent that in dogs.
• the polypeptides of the vaccine composition according to the invention can be inoculated at doses of between 1 and 500 g, preferably between 10 and 100 g (Wressnigg et al., 2013 [16]).
• the synthesis of the polypeptides that can be used in the vaccine composition according to the present invention can be carried out by any method known to those skilled in the art. It can be for example a synthesis by genetic engineering.
• polypeptides of the vaccine composition according to the invention When the synthesis of the polypeptides of the vaccine composition according to the invention is carried out by genetic engineering, it is possible, for example, to construct a large polypeptide comprising the polypeptide of the vaccine composition of the present invention and to digest it with restriction enzymes in order to recovering said polypeptide from the vaccine composition according to the invention.
• restriction enzymes for example, the protocol described in F. Cordier-Ochsenbein et al. J. Mol. Biol. 279.1 177-1 185 [17].
• the vaccine composition according to the invention can be produced according to any method well known to those skilled in the art. It may be for example a simple mixture of the various components of the vaccine composition.
• the document by Ramamoorthi and Smooker (2009) [18] describes a method of manufacturing a vaccine composition that can be used in the context of the present invention.
• the present invention provides effective solutions for the prevention of Lyme disease.
• FIG. 1 represents the PCR quantification of B. burgdorferi, native strain 297 and its virulent clone 297c4, in the skin of mice, with respect to the time after inoculation of the strain.
• N Fia / 10 4 GAPDH means number of flagellin per 10 4 of Glyceraldehyde 3-phosphate dehydrogenase.
• FIG. 2 represents an expression profile by RT-PCR of a protein common to the three species of Borrelia: B. burgdorferi ss, B. afzelii and B. garinii, namely BB0566 (SEQ ID NO: 10) in the Mouse skin during the early transmission of the bacteria.
• Example 1 Strategy for identifying Borrelia vaccine candidates for Lyme disease
• the inventors have developed a proteomic approach to identify and select effective polypeptides for the prevention of Lyme disease.
• CSF cerebrospinal fluid
• Bacterial clones were selected for their virulence in mice. All strains were grown in complete BSK-H (Sigma) at 33 ° C and used at low passage ( ⁇ 7). Borrelia were counted and viability was verified by dark field microscopy.
• the proteins were extracted with a Laemmli buffer [20]. After sonication and centrifugation, the pellet was removed and the protein concentration of the supernatant was determined. Proteins (75 g) underwent a one-dimensional gel electrophoresis prefractionation step (electrophoresis) SDS-PAGE
• NanoLC-MS / MS analysis was performed using a nanoLC-Chip / MS system (Agilent Technologies, Palo Alto, CA) coupled to an amaZon ion trap (Bruker, Bremen, Germany).
• the chromatographic system consisted of a pre-column (40 nL, 5 ⁇ ) and a column (150 mm x 75 ⁇ , 5 ⁇ ) comprising the same Zorbax stationary phase 300SB-C18.
• the solvent system consisted of 2% ACN, 0.1% HCO 2 H in water (solvent A) and 2% water, 0.1% HCO 2 H in ACN (solvent B).
• 1 ⁇ l of peptide extract (1/20 of the total volume) was loaded in duplicate on the pre-column (in the enrichment column) at a loading rate of (a flow rate set at) 3.75 ⁇ / min with solvent A (100% solvent A).
• the elution was carried out at a flow rate of 300 nl / min by application of a linear gradient of 8-40% of solvent B for 30 minutes followed by a step of 4 min at 70% of solvent B before reconditioning. the 8% solvent column B.
• the acquisition parameters of the MS and MS / MS spectra are as follows: source temperature set at 145 ° C. and gas flow rate at 4 L / min.
• the voltage applied to the needle of the sprayer was set at -1900 V.
• the acquisition of the MS spectra was performed in positive (ions) mode over a mass range of 250 to 1500 m / z at a scanning speed of 8100 m / z per sec.
• the maximum number of ions (ionic charge control) and the maximum accumulation time were respectively set at 200,000 and 200 ms, with an average of two scans.
• MS / MS spectra were acquired by sequentially selecting the 8 most intense (abundant) precursor ions, with a preference for the dicharged ions.
• the ion selection threshold for fragmentation was set at 100,000. Fragmentation was performed using argon as the collision gas. The selected ions were excluded for 0.6 min.
• the MS / MS spectra were carried out over a mass range ranging from 100 to 2000 m / z.
• the maximum number of ions accumulated in MS / MS (control ionic charge) was set at 400,000, with an average of 5 scans.
• the complete system was driven by the Hystar 3.2 (Bruker) software.
• the number of spectra attributed to each protein in each duplicate was used to detect expressed in virulent clones.
• the beta-binomial assay [25] was performed at R to determine protein overexpression (p ⁇ 0.05) in each virulent clone relative to the wild-type strain. The test was performed independently for each of the search engines because the spectral identifications are dependent on the algorithms.
• the homologous proteins in the three species were determined using the blastp program [27] with an E-value threshold set at 10-30 . Three proteins are thus common to the three Borrelia species analyzed and 27 proteins are common to at least two of the three species (see Table 1 above).
• BB0173 SEQ ID NO: 13
• VWA von Willebrand factor A
• BB0566 SEQ ID NO: 10
• STAS "Sulfate Transporter and Anti-sigma factor antagonist” type domain
• BAPKO0873 (SEQ ID NO: 59) contains a domain ⁇ of an RNA polymerase (RpoZ) subunit.
• BB0765 (SEQ ID NO: 27) contains a domain III of the DNA polymerase (DNAX).
• RpoN directly activates the transcription of RpoS which, in turn, controls the expression of membrane-associated lipoproteins associated with virulence (OspA, OspC, decorin-binding proteins).
• OspA membrane-associated lipoproteins associated with virulence
• a protein associated with an EBfC nucleotide appears as a global regulator of gene expression in Borrelia. The increase in EBfC levels influences the expression of B. burgdorferi genes by 4.5%, including genes associated with infection.
• Other proteins involved in DNA replication, recombination and repair DNA helicase and SBCD exonuclease
• SBCD exonuclease DNA helicase and SBCD exonuclease
• periplasmic flagella FliE, FliP, FlgE and flagellum-specific ATP synthase (FIN).
• Mobility is crucial for the infectious cycle of B. burgdorferi and periplasmic flagella is essential to ensure adequate mobility to bacteria. Inactivation of genes coding for flagella proteins has been shown to lead to non-motile bacteria. A Another study showed that flagella loss decreases B. garinii infection.
• PTS phosphotransferase-related protein
• ACP acyl-bearing protein
• the skin is an essential organ in the development of Lyme borreliosis because Borrelia is inoculated and multiplies before spreading in the body to reach the target organs: the joint, the nervous system and the skin at a distance.
• mice were infected with 10 3 spirochaetes in 0.1 ml of BSK medium intradermally in the dorsolumbar region.
• the control mice were injected with an equal volume of sterile BSK medium and maintained under the same conditions as the infected animals.
• Evaluation of arthritis was performed weekly by measuring the thickness of both tibio-tarsal joints with a metric vernier caliper. Joint measures provided an indication of the severity of arthritis. Serology was performed as described in Kern et al. [29].
• mice were killed by overdose of isoflurane gas. About 1 cm of skin was collected at the site of inoculation and stored in Trizol (registered trademark) (Invitrogen).
• mice The ear, the base of the heart, the bladder and the tibiotarsal joints of Each mouse was aseptically collected and divided into two parts, for PCR and Borrelia culture. The organs of the uninfected mice were collected under the same conditions as the positive mice.
• spirochaetes For the detection of spirochaetes by culture, the organs removed were placed in 6 ml of BSK-H medium containing 30 g of rifampicin (BioRad). The tubes were maintained at 33 ° C, and the presence of spirochaetes was examined weekly by darkfield microscopy.
• DNA was extracted from the organs of each mouse on a MagNA Pure system (Roche Diagnostics, France), using a MagNA Pure LC wide-volume isolation kit after external lysis.
• the heart, the bladder, the ear and the skin were placed in 500 ⁇ l of lysis buffer containing proteinase K.
• Other samples were treated with external lysis by collagenase A and then proteinase K.
• the DNA samples were finally eluted in 100 ⁇ l of elution buffer.
• Ten ⁇ of Borrelia DNA was used as a positive control for detection.
• Qualitative amplification was performed as described in Woods et al. [30]., Targeting the flagellin gene.
• RNA samples were taken from each mouse at the site of inoculation.
• Total RNA was purified using Trizol reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNAs were determined by measuring the optical density at A260 and A280. The samples were then treated with gDNAse (QIAGEN) to remove the contamination with DNA. Total extracted RNAs were submitted to the Quantiscript Reverse Transcription (QIAGEN) to produce the cDNA.
• CDNA was used to quantify the ospC and bbk32 genes. For B. burgdorferi C297 / 4, selected genes corresponding to cell envelope proteins were retained for RT-PCR.
• Relative expression levels were calculated using the AACt method with flagellin as the internal standard.
• the amplification and detection were performed with an ABI 7500 system with the following thermal profile: 95 ° C for 10 minutes, 50 cycles of 95 ° C for 15 s, at 50 ° C for 30 s and 60 ° C for 1 min.
• Each amplification condition was compared to day 3 for relative quantization.
• Correlation factors were calculated by comparing the cDNA amplification of each kinetic point of the native strain to the cDNA amplification of each point of the hypervirulent clone. Then, the curve obtained for the clone, was normalized by these factors to obtain a second curve, representative of the wild-type strain, and quantitatively comparable to the clone.
• the inflammatory profile in the skin of the mice was compared for these different strains of B. burgdorferi ss.
• Antimicrobial peptides AMPs
• the PBre (EM) strain induced a significant amount of cathelicidin with a peak on day 3.
• the MR726 (MEM) strain strongly induced defensin mBD-3.
• Wild type strain 297 (CSF) showed a peak of mBD-3 at 24 h while strain 1808/03 (CSF) induced a negligible amount of all three MPAs tested.
• the induction of additional pro-inflammatory molecules was then measured: TNF- ⁇ , IL-6, IL-22 and the chemokine MCP-1.
• Borrelia infection could be initiated by a heterogeneous population of Borrelia in the vertebrate host.
• a clone C297 / 4 from B. burgdorferi ss 297 was selected in the laboratory for its rapid diffusion and its neurological manifestations in mice [31].
• the virulent clone C297 / 4 caused inflammation of the skin with a greater induction of defensins, MBD-14, and cathelicidin compared to the wild-type strain.
• the results of the wild strain 297 and the hypervirulent clone were also compared in the C3H / HeN mouse.
• the hypervirulent clone spread more rapidly to the joint, while diffusion to other organs was similar to that of the wild-type strain.
• Quantification of the bacterial load in the tissues confirmed the intense multiplication occurring in the skin at day 7 regardless of the strain used, but no significant difference was observed between the hypervirulent clone and the wild strain 297.
• mice Five proteins were selected for in vivo tests in mice, namely the three "hypothetical protein" proteins only detected in the virulent clones and common to the three species of Borrelia (SEQ ID NO: 10 (BB0566), 13 (BB0173) and 16 (BB0722) of Borrelia burgdorferi ss and corresponding sequences SEQ ID NO: 11 (BAPKO0596), 14 (BAPKO0175) and 17 (BAPKO0766) respectively of B. afzelii) and 12 (BG0576), (BG0172) and 18 (BG0172). BG0744) of B.
• mice Three to four week old C3H / HeN mice were purchased from Charles River Laboratories (L'ArbresIe, France).
• the inventors have been particularly interested in the strain B. burgdorferi ss 297, isolated from cerebrospinal fluid in the United States.
• mice All Borrelia strains were cultured in BSK-H (Sigma) medium at 33 ° C and used at low passage ( ⁇ 7) for mouse infection. Spirochetes were counted and viability was verified using the dark-field microscope. The mice were infected with 10 3 spirochaetes in 0.1 ml BSK by intradermal injection into the thoracic dorsal region.
• mice were killed by isoflurane.
• a 1 cm area of mouse skin was collected at the site of inoculation and stored in Trizol (Invitrogen) for RT-PCR analyzes.
• Trizol Invitrogen
• the sample is kept dry at -80 ° C.
• RNA samples were taken from each mouse at the site of inoculation.
• Total RNA was purified using Trizol reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNA was determined by measuring A260 and A280. The samples were then treated with gDNAse wipeout (QIAGEN).
• Total RNA extracted was synthesized into cDNA using Quantiscript Reverse Transcription (QIAGEN). CDNA was used to quantify the bbk32 genes (positive control). For B. burgdorferi ss 297 and 297c4, the genes corresponding to the three common proteins and RpoN and Gnd were tested in RT-PCR using the primers described in Table 3 below.
• Biopsies were selected based on PCR quantification. Fragments of approximately 4 mg were cut out and the proteins were extracted into 200 ⁇ l Laemmli buffer and then assayed. Proteins (50 g) were pre-fractionated on SDS-PAGE electrophoresis gel and the migration tracks were excised and processed as described in Example 1. The tryptic peptides were analyzed by nanoLC-MS / MS using the nanoLC-Chip / MS system coupled to the amaZon ion trap, as described in Example 1. The spectra of MS and MS / MS were acquired with the same parameters and the searches were carried out in the same way except for the databanks. In this case, the searches were carried out in databases, consisting of sequences of B. burgdorferi ss B31 and mice, downloaded from the database NCBInr and UniProtKB-SwissProt, respectively (B. burgdorferi B31: August 16 2012; mouse: April 19, 2013).
• the RT-PCR expression profile of the protein BB0566 (SEQ ID NO: 10), a protein common to all three Borrelia species: B. burgdorferi ss, B. afzelii and B. garinii, in the skin of mice during the early transmission of the bacteria is shown in Figure 2.
• mice In cutaneous biopsies of infected mice, an average of 1350 mouse proteins were identified. Among the Borrelia proteins detected, the RpoN and Gnd proteins were identified which confirms the expression of these proteins in the skin seven days after inoculation and their potential role during the early transmission of the bacterium.
• the dose of recombinant proteins to be administered is determined according to a prior dose-effect study well known to those skilled in the art, generally between 1 and 500 g. Depending on the vaccination protocol in the dog, ideally, two administrations will be performed between 2 to 4 weeks apart and an annual reminder.
• the adjuvant is chosen according to its ability to stimulate the humoral response and / or the cellular response. The person skilled in the art knows which adjuvant to choose to effectively stimulate the humoral response and / or the cellular response.
• the administration of the vaccine is carried out intradermally, subcutaneously or intramuscularly, preferably intramuscularly or subcutaneously. List of references

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