Patent Publication Number: US-2016220653-A1

Title: Lyme disease vaccines

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This is a U.S. national stage of application No. PCT/FR2014/052085, filed on 12 Aug. 2014. Priority under 35 U.S.C. §119(a) and 35 U.S.C. §365(b) is claimed from French Application No. 13/58017 filed on 14 Aug. 2013, the disclosure of which is also incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to Lyme disease vaccines, in particular to vaccines comprising one or more isolated polypeptides of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii.    
     The present invention has applications in the veterinary and medical fields. 
     In the description below, the references between square brackets ([ ]) refer to the list of references presented at the end of the examples. 
     BACKGROUND 
     Lyme borreliosis, also known as Lyme disease, is a vector-transmitted disease transmitted by a hard tick of the  Ixodes  genus. It is rife mainly in the Northern hemisphere where it constitutes the most common vector-transmitted disease. Recent data hint at its area of distribution also extending into the Southern hemisphere with human cases in Australia (Mayne et al., 2011 [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  Borrelia burgdorferi  sensu lato group with approximately 20 species identified. 
     Lyme borreliosis usually develops in the wild fauna in a wide range of vertebrate hosts and manifests itself accidentally in humans first through a skin inflammation, erythema migrans, and then through very varied clinical manifestations: joint, cardiac, neurological and skin manifestations (Radolf et al., 2012 [3]; Stanek et al., 2012 [4]). The skin therefore constitutes an essential interface in the transmission during the tick bite and the development of the disease. The clinical symptoms of borreliosis observed in dogs are very similar to those observed in humans, but more specifically it induces glomerulonephritis in dogs (Little et al., 2010 [5]). 
     Although antibiotic treatments are effective at an early stage of the infection, many patients develop borreliosis because of an absence or a non-observation of erythema migrans, or because of treatment which is inappropriate or too late. A vaccine approach appears to be more promising for treating or preventing Lyme disease, in particular in animals in which the early skin stage cannot be observed owing to the coat. 
     There are currently several vaccines on the market for preventing canine borreliosis. In the United States, they are directed only against a single species,  Borrelia burgdorferi  sensu stricto. Moreover, based on the concept of the “transmission blocking vaccine”, a vaccine was marketed in the United States for humans, LYMErix (trademark), but the marketing of said vaccine was stopped in 2002 following side effects in certain patients (Hanson and Edelman, 2003 [6]). Using a recombinant antigen, OspA (Outer surface protein A), two vaccines are currently used in dogs in the United States (Nobivac (registered trademark) sold by Intervet and Recombitek (registered trademark) sold by Merial) and have shown a certain amount of efficacy, but only against the species  B. burgdorferi  ss (Lafleur et al., 2009 [7]). Indeed, the  Borrelia  population transmitted by the ticks is very heterogeneous in Europe, and the vaccines currently on the market are not effective against the other virulent species of  Borrelia  which are predominant in Europe. A third vaccine is sold using a bacterial lysate of  B. burgdorferi  ss (Fort Dodge). In Europe, only two companies (Merial and Bioveta) sell a vaccine also based on lysates of  Borrelia, B. burgdorferi  ss for Merial and  B. afzelii  and  B. garinii  for Bioveta. For these various vaccines, three injections are generally required in order to obtain sufficient protection, generally controlled by measuring the antibodies by ELISA (Topfer and Straubinger, 2007 [8]). However, the use of bacterial lysate as a vaccine base is not satisfactory for mass production. 
     Thus, the existing vaccines use either bacterial lysates of which the mass production is difficult to carry out, or the recombinant protein OspA, which is not very immunogenic and not very highly expressed at the beginning of the infection in humans, or OspC, the proof of concept of which from a vaccine point of view has not been established. 
     There are therefore real needs to develop novel vaccines which overcome these defects, drawbacks and obstacles of the prior art, in particular vaccines which are strongly immunogenic and effective on various  Borrelia  populations, and which can be mass produced at low cost. 
     SUMMARY 
     The present invention precisely meets the abovementioned needs of the prior art, by providing vaccine compositions for the prevention of Lyme disease. 
     For this purpose, the inventors have developed a proteomic approach in order to identify and select polypeptides which are effective for preventing Lyme disease. This approach has been carried out on the basis of three  Borrelia  species that the inventors have determined as being the most involved in human and animal pathology, in particular in dogs, namely  Borrelia burgdorferi  ss,  Borrelia afzelii  and  Borrelia garinii.    
     Thus, a subject of the present invention is in particular a vaccine composition comprising at least one polypeptide of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii , chosen from the sequences described hereinafter. 
     DETAILED DESCRIPTION 
     In particular, the sequences SEQ ID NOs: 1 to 92 are described, presented in table 1 below, in which appear the numbers of sequences of the appended sequence listing, the names of the corresponding polypeptides and the names of the corresponding loci in the genome of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii.    
     In other words, table 1 describes the isolated polypeptides consisting of a sequence chosen from SEQ ID NOs: 1 to 92 which are likely to be used in the vaccine composition of the invention. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequences of the polypeptides that can be used in a vaccine 
               
               
                 composition according to the invention 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Name of 
                   
                   
                   
                 Name of 
               
               
                   
                 SEQ 
                 the locus in 
                 SEQ 
                 Name of the 
                 SEQ 
                 the 
               
               
                   
                 ID 
                 
                   B. burgdorferi 
                 
                 ID 
                 locus in 
                 ID 
                 locus in 
               
               
                 Polypeptide name 
                 NO 
                 ss 
                 NO 
                 
                   B. afzelii 
                 
                 NO 
                 
                   B. garinii 
                 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 n/a 
                 1 
                 BB0213 
                 — 
                 — 
                 — 
                 — 
               
               
                 UDP-N- 
                 2 
                 BB0304 
                 — 
                 — 
                 — 
                 — 
               
               
                 acetylmuramoylalanyl- 
               
               
                 D-glutamyl-2,6- 
               
               
                 diaminopimelate--D- 
               
               
                 alanyl-D-alanine ligase 
               
               
                 (murF) 
               
               
                 fibronectin/fibrinogen- 
                 3 
                 BB0347 
                 — 
                 — 
                 — 
                 — 
               
               
                 binding protein, 
               
               
                 putative 
               
               
                 penicillin-binding 
                 4 
                 BB0718 
                 — 
                 — 
                 — 
                 — 
               
               
                 protein (pbp-2) 
               
               
                 n/a 
                 5 
                 BB0761 
                 — 
                 — 
                 — 
                 — 
               
               
                 n/a 
                 6 
                 BB0823 
                 — 
                 — 
                 — 
                 — 
               
               
                 outer membrane 
                 7 
                 BB0167 
                 — 
                 — 
                 — 
                 — 
               
               
                 protein (tnp50) 
               
               
                 alanine racemase (alr) 
                 8 
                 BB0160 
                 — 
                 — 
                 — 
                 — 
               
               
                 hemolysin III (yplQ) 
                 9 
                 BB0117 
                 — 
                 — 
                 — 
                 — 
               
               
                 n/a 
                 10 
                 BB0566 
                 11 
                 BAPKO0596 
                 12 
                 BG0576 
               
               
                 n/a 
                 13 
                 BB0173 
                 14 
                 BAPKO0175 
                 15 
                 BG0172 
               
               
                 n/a 
                 16 
                 BB0722 
                 17 
                 BAPKO0766 
                 18 
                 BG0744 
               
               
                 50 S ribosomal protein 
                 19 
                 BB0229 
                 — 
                 — 
                 20 
                 BG0232 
               
               
                 L31 type B 
               
               
                 flagellar hook-basal 
                 21 
                 BB0292 
                 — 
                 — 
                 22 
                 BG0295 
               
               
                 body protein FliE 
               
               
                 Beta glucosidase, 
                 23 
                 BB0620 
                 — 
                 — 
                 24 
                 BG0639 
               
               
                 putative 
               
               
                 30S ribosomal protein 
                 25 
                 BB0491 
                 — 
                 — 
                 26 
                 BG0503 
               
               
                 S14 type Z 
               
               
                 n/a 
                 27 
                 BB0765 
                 — 
                 — 
                 28 
                 BG0788 
               
               
                 n/a 
                 29 
                 BB0748 
                 30 
                 BAPKO0794 
                 — 
                 — 
               
               
                 Preprotein translocase 
                 31 
                 BB0395 
                 32 
                 BAPKO0410 
                 — 
                 — 
               
               
                 subunit SecE 
               
               
                 Holo ACP synthase 
                 33 
                 BB0010 
                 34 
                 BAPKO009 
                 — 
                 — 
               
               
                 n/a 
                 35 
                 BB0029 
                 36 
                 BAPKO028 
                 — 
                 — 
               
               
                 n/a 
                 37 
                 BB0081 
                 38 
                 BAPKO0081 
                 — 
                 — 
               
               
                 n/a 
                 39 
                 BB0102 
                 40 
                 BAPKO0103 
                 — 
                 — 
               
               
                 RNA polymerase 
                 41 
                 BB0450 
                 42 
                 BAPKO0472 
                 — 
                 — 
               
               
                 sigma-54 factor 
               
               
                 Type III pantothenate 
                 43 
                 BB0527 
                 44 
                 BAPKO0553 
                 — 
                 — 
               
               
                 kinase 
               
               
                 tRNA (guanine-N(1)-)- 
                 45 
                 BB0698 
                 46 
                 BAPKO0742 
                 — 
                 — 
               
               
                 methyltransferase 
               
               
                 n/a 
                 — 
                 — 
                 47 
                 BAPKO0189 
                 48 
                 BG0186 
               
               
                 n/a 
                 — 
                 — 
                 49 
                 BAPKO0265 
                 50 
                 BG0258 
               
               
                 flagellar biosynthesis 
                 — 
                 — 
                 51 
                 BAPKO0285 
                 52 
                 BG0278 
               
               
                 protein FliP 
               
               
                 Nucleoid associated 
                 — 
                 — 
                 53 
                 BAPKO0491 
                 54 
                 BG0475 
               
               
                 protein EbfC 
               
               
                 Acyl carrier porter 
                 — 
                 — 
                 55 
                 BAPKO0747 
                 56 
                 BG0726 
               
               
                 (ACP) 
               
               
                 n/a 
                 — 
                 — 
                 57 
                 BAPKO0861 
                 58 
                 BG0834 
               
               
                 n/a 
                 — 
                 — 
                 59 
                 BAPKO0873 
                 60 
                 BG0845 
               
               
                 n/a 
                 — 
                 — 
                 61 
                 BAPKO4502 
                 62 
                 BGP215 
               
               
                 n/a 
                 — 
                 — 
                 63 
                 BAPKO0132 
                 64 
                 BG0132 
               
               
                 n/a 
                 — 
                 — 
                 65 
                 BAPKO0215 
                 66 
                 BG0210 
               
               
                 tRNA dimethylallyl- 
                 — 
                 — 
                 67 
                 BAPKO0874 
                 68 
                 BG0846 
               
               
                 transferase 
               
               
                 exonuclease SbcD 
                 — 
                 — 
                 69 
                 BAPKO0882 
                 70 
                 BG0854 
               
               
                 Hypothetical protein 
                 — 
                 — 
                 71 
                 BAPKO0366 
                 72 
                 BG0366 
               
               
                 RNA polymerase 
                 73 
                 BB0388 
                 74 
                 BAPKO0029 
                 — 
                 — 
               
               
                 subunit beta 
               
               
                 replicative DNA 
                 75 
                 BB0111 
                 76 
                 BAPKO0112 
                 — 
                 — 
               
               
                 helicase 
               
               
                 6-phosphogluconate 
                 77 
                 BB0561 
                 78 
                 BAPKO0590 
                 — 
                 — 
               
               
                 dehydrogenase 
               
               
                 flagellum-specific ATP 
                 79 
                 BB0288 
                 80 
                 BAPKO0298 
                 — 
                 — 
               
               
                 synthase FliI 
               
               
                 putative 
                 81 
                 BB0627 
                 82 
                 BAPKO0669 
                 — 
                 — 
               
               
                 aminopeptidase 2 
               
               
                 flagellar hook protein 
                 83 
                 BB0283 
                 84 
                 BAPKO0293 
                 — 
                 — 
               
               
                 FlgE 
               
               
                 phosphofructokinase 
                 85 
                 BB0727 
                 86 
                 BAPKO0771 
                 — 
                 — 
               
               
                 5- 
                 — 
                 — 
                 87 
                 BAPKO0619 
                 88 
                 BG0601 
               
               
                 methylthioadenosine/S- 
               
               
                 adenosylhomocysteine 
               
               
                 nucleosidase, putative 
               
               
                 n/a 
                 — 
                 — 
                 89 
                 BAPKO0034 
                 90 
                 BG0034 
               
               
                 PTS system, maltose 
                 — 
                 — 
                 91 
                 BAPKO0027 
                 92 
                 BGB26 
               
               
                 and glucose-specific 
               
               
                 IIABC component 
               
               
                   
               
            
           
         
       
     
     In the present invention, unless otherwise indicated, “ Borrelia burgdorferi ” signifies “ Borrelia burgdorferi  ss” or “ Borrelia burgdorferi  sensu stricto”, as opposed to the indication “ Borrelia burgdorferi  sensu lato” which covers approximately 20 different species. 
     In addition, unless otherwise indicated, the letters determining the sequences of the polypeptides hereby described correspond to the one-letter abbreviation proposed by Leder (Leder et al. Introduction to molecular medicine, Ed Scientific American, 1994 [9]). 
     Thus, a subject of the present invention is in particular a vaccine composition comprising at least one polypeptide of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii  chosen from the sequences SEQ ID NO: 1 to 92. Preferably, the at least one polypeptide is chosen from the sequences SEQ ID NOs: 10 to 92, preferably 10 to 18, preferably 10 to 15. 
     Any polypeptide of sequence SEQ ID NOs: 1 to 92, or any combination of at least two of the polypeptides of sequences SEQ ID NOs: 1 to 92, can be used in the vaccine composition according to the invention. 
     For example, the combination of polypeptides may comprise 2 different polypeptides of sequence SEQ ID NOs: 1 to 92, for example 3, 4, 5, 6, 7, 8, 9 or even more than 9 different polypeptides of sequence SEQ ID NOs: 1 to 92. A combination of the different polypeptides of sequence SEQ ID NOs: 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. For example, the vaccine composition according to the invention may comprise a combination of two polypeptides as presented in table 2 below. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Combination of polypeptides in the vaccine composition 
               
               
                 according to the invention 
               
            
           
           
               
               
            
               
                 Polypeptide 
                   
               
               
                 of sequence 
                 Possible combination with the polypeptides of sequence  
               
               
                 SEQ ID NO: 
                 SEQ ID NO: as follows 
               
               
                   
               
            
           
           
               
               
            
               
                 1 
                 At least one from: 2 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 2 
                 At least one from: 1 and 3 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 3 
                 At least one from: 1, 2 and 4 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 4 
                 At least one from: 1 to 3 and 5 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 5 
                 At least one from: 1 to 4 and 6 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 6 
                 At least one from: 1 to 5 and 7 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 7 
                 At least one from: 1 to 6 and 8 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 8 
                 At least one from: 1 to 7 and 9 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 9 
                 At least one from: 1 to 8 and 10 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18, 47 to 72 and 87 to 92 
               
               
                 10 
                 At least one from: 1 to 9 and 11 to 92,  
               
               
                   
                 preferably at least one from: 13 to 18 
               
               
                 11 
                 At least one from: 1 to 10 and 12 to 92,  
               
               
                   
                 preferably at least one from: 13 to 18 
               
               
                 12 
                 At least one from: 1 to 11 and 13 to 92,  
               
               
                   
                 preferably at least one from: 13 to 18 
               
               
                 13 
                 At least one from: 1 to 12 and 14 to 92,  
               
               
                   
                 preferably at least one from: 10 to 12 and 16 to 18 
               
               
                 14 
                 At least one from: 1 to 13 and 15 to 92,  
               
               
                   
                 preferably at least one from: 10 to 12 and 16 to 18 
               
               
                 15 
                 At least one from: 1 to 14 and 16 to 92,  
               
               
                   
                 preferably at least one from: 10 to 12 and 16 to 18 
               
               
                 16 
                 At least one from: 1 to 15 and 17 to 92,  
               
               
                   
                 preferably at least one from: 10 to 15 
               
               
                 17 
                 At least one from: 1 to 16 and 18 to 92,  
               
               
                   
                 preferably at least one from: 10 to 15 
               
               
                 18 
                 At least one from: 1 to 17 and 19 to 92,  
               
               
                   
                 preferably at least one from: 10 to 15 
               
               
                 19 
                 At least one from: 1 to 18 and 20 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 20 
                 At least one from: 1 to 19 and 21 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 21 
                 At least one from: 1 to 20 and 22 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 22 
                 At least one from: 1 to 21 and 23 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 23 
                 At least one from: 1 to 22 and 24 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 24 
                 At least one from: 1 to 23 and 25 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 25 
                 At least one from: 1 to 24 and 26 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 26 
                 At least one from: 1 to 25 and 27 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 27 
                 At least one from: 1 to 26 and 28 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 28 
                 At least one from: 1 to 27 and 29 to 92,  
               
               
                   
                 preferably at least one from: 10 to 18 and 29 to 92 
               
               
                 29 
                 At least one from: 1 to 28 and 30 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 30 
                 At least one from: 1 to 29 and 31 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 31 
                 At least one from: 1 to 30 and 32 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 32 
                 At least one from: 1 to 31 and 33 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 33 
                 At least one from: 1 to 32 and 34 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 34 
                 At least one from: 1 to 33 and 35 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 35 
                 At least one from: 1 to 34 and 36 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 36 
                 At least one from: 1 to 35 and 37 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 37 
                 At least one from: 1 to 36 and 38 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 38 
                 At least one from: 1 to 37 and 39 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 39 
                 At least one from: 1 to 38 and 40 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 40 
                 At least one from: 1 to 39 and 41 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 41 
                 At least one from: 1 to 40 and 42 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 42 
                 At least one from: 1 to 41 and 43 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 43 
                 At least one from: 1 to 42 and 44 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 44 
                 At least one from: 1 to 43 and 45 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 45 
                 At least one from: 1 to 44 and 46 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 46 
                 At least one from: 1 to 45 and 47 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 47 
                 At least one from: 1 to 46 and 48 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 48 
                 At least one from: 1 to 47 and 49 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 49 
                 At least one from: 1 to 48 and 50 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 50 
                 At least one from: 1 to 49 and 51 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 51 
                 At least one from: 1 to 50 and 52 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 52 
                 At least one from: 1 to 51 and 53 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 53 
                 At least one from: 1 to 52 and 54 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 54 
                 At least one from: 1 to 53 and 55 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 55 
                 At least one from: 1 to 54 and 56 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 56 
                 At least one from: 1 to 55 and 57 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 57 
                 At least one from: 1 to 56 and 58 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 58 
                 At least one from: 1 to 57 and 59 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 59 
                 At least one from: 1 to 58 and 60 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 60 
                 At least one from: 1 to 59 and 61 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 61 
                 At least one from: 1 to 60 and 62 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 62 
                 At least one from: 1 to 61 and 63 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 63 
                 At least one from: 1 to 62 and 64 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 64 
                 At least one from: 1 to 63 and 65 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 65 
                 At least one from: 1 to 64 and 66 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 66 
                 At least one from: 1 to 65 and 67 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 67 
                 At least one from: 1 to 66 and 68 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 68 
                 At least one from: 1 to 67 and 69 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 69 
                 At least one from: 1 to 68 and 70 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 70 
                 At least one from: 1 to 69 and 71 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 71 
                 At least one from: 1 to 70 and 72 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 72 
                 At least one from: 1 to 71 and 73 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 73 
                 At least one from: 1 to 72 and 74 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 74 
                 At least one from: 1 to 73 and 75 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 75 
                 At least one from: 1 to 74 and 76 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 76 
                 At least one from: 1 to 75 and 77 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 77 
                 At least one from: 1 to 76 and 78 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 78 
                 At least one from: 1 to 77 and 79 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 79 
                 At least one from: 1 to 78 and 80 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 80 
                 At least one from: 1 to 79 and 81 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 81 
                 At least one from: 1 to 80 and 82 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 82 
                 At least one from: 1 to 81 and 83 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 83 
                 At least one from: 1 to 82 and 84 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 84 
                 At least one from: 1 to 83 and 85 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 85 
                 At least one from: 1 to 84 and 86 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 86 
                 At least one from: 1 to 85 and 87 to 92,  
               
               
                   
                 preferably at least one from: 10 to 28, 47 to 72 and 87 to 92 
               
               
                 87 
                 At least one from: 1 to 86 and 88 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 88 
                 At least one from: 1 to 87 and 89 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 89 
                 At least one from: 1 to 88 and 90 to 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 90 
                 At least one from: 1 to 89, 91 and 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 91 
                 At least one from: 1 to 90 and 92,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                 92 
                 At least one from: 1 to 91,  
               
               
                   
                 preferably at least one from: 1 to 46 and 73 to 86 
               
               
                   
               
            
           
         
       
     
     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 than those of the combination, or even a fourth different polypeptide, etc. Preferably, the vaccine composition comprises a combination of 2, 3, 4, 5, 6 or 7 different polypeptides. 
     The 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 NOs: 1 to 92. Those skilled in the art clearly understand that sequences exhibiting a homology or an identity with these sequences can also be used, in an equivalent manner, in the vaccine composition according to the invention, provided that they have the same effect as the polypeptides of sequence SEQ ID NOs: 1 to 92, namely an immunogenic effect, of use 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. For example, a sequence used can have greater than 80% homology or identity with a sequence described in table 1, for example greater than 85%, or than 90%, or than 95%, or than 99% identity or homology 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, for example, be the BLAST (Basic Local Alignment Search Tool) method described in the document Altschul, S. F. et al., J. Mol. Biol. 1990 [27]. 
     In table 1 above, the polypeptide sequences which appear on the same line represent the same protein, the name of which is indicated in the left-hand column. “n/a” signifies that the name of the protein in question has not been identified or is not yet known. 
     Although the polypeptides which appear on the same line represent the same protein, their amino acid sequences are not strictly identical. This may be due to the possible mutations which have occurred distinctly in the various species of the  Borrelia  genus. By way of examples, the sequences SEQ ID NOs: 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 chosen from  Borrelia burgdorferi  ss,  Borrelia afzelii  and  Borrelia garinii . For example:
         the polypeptides of sequence SEQ ID NOs: 1 to 9 are proteins specific to the virulent clone of  Borrelia burgdorferi  ss;   the polypeptides of sequence SEQ ID NOs: 10 to 18 are proteins common to the virulent clones of  Borrelia burgdorferi  ss,  Borrelia afzelii  and  Borrelia garinii;      the polypeptides of sequence SEQ ID NOs: 19 to 28 are proteins common to the virulent clones of  Borrelia burgdorferi  ss and  Borrelia garinii,      the polypeptides of sequence SEQ ID NOs: 29 to 46 are proteins common to the virulent clones of  Borrelia burgdorferi  ss and  Borrelia afzelii;      the polypeptides of sequence SEQ ID NOs: 47 to 72 are proteins common to the two virulent clones of  Borrelia afzelii  and  Borrelia      the polypeptides of sequence SEQ ID NOs: 73 to 86 are proteins common to  Borrelia burgdorferi  ss and  Borrelia afzelii ; and   the polypeptides of sequence SEQ ID NOs: 87 to 92 are proteins common to  Borrelia afzelii  and  Borrelia garinii.          

     The polypeptides of sequence SEQ ID NOs: 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.    
     Advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NOs: 1 to 92, the polypeptides are polypeptides of different sequences. Likewise advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NOs: 1 to 92, the polypeptides represent different proteins. 
     Advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NOs: 1 to 92, the polypeptides may be proteins the combination of which makes it possible to obtain an immunization simultaneously against two of the species or the three species  Borrelia burgdorferi  ss,  Borrelia afzelii  and  Borrelia garinii . In other words, advantageously, the composition according to the invention comprises a protein common to the abovementioned three species or a mixture of several proteins, for example, 2, 3 or 4 proteins, or even more, covering these three species. This embodiment makes it possible to provide vaccine compositions which are universal with respect to the  Borrelia  populations. 
     According to one particular embodiment, 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 NOs: 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. Preferably, the at least one polypeptide is chosen from SEQ ID NOs: 10 to 15. 
     In this embodiment, the vaccine composition may also comprise at least one other polypeptide of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii  chosen from SEQ ID NOs: 1, 3 to 6, 16 to 18, 21 to 24, 27, 28, 31, 32, 37 to 40, 51, 52, 55, 56 and 73 to 84. 
     In addition, in this embodiment, the vaccine composition may also comprise at least one other polypeptide of  Borrelia burgdorferi, Borrelia afzelii  or  Borrelia garinii  chosen from the groups (c1), (c2) and (c3), 
     said group (c1) comprising SEQ ID NOs: 19 to 28, 
     said group (c2) comprising SEQ ID NOs: 29 to 46 and 73 to 86, 
     said group (c3) comprising SEQ ID NOs: 47 to 72 and 87 to 92, 
     on the condition that, if said at least one polypeptide chosen from SEQ ID NOs: 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:
         is included in one of the groups (c1), (c2) or (c3), said at least one other polypeptide is included in a different group (c1), (c2) or (c3), or   is SEQ ID NO: 2, 7, 8 or 9, said at least one other polypeptide is included in the group (c3), or   is SEQ ID NO: 10, 11, 12, 13, 14 or 15, said at least one other polypeptide is included in any of the groups (c1), (c2) and (c3).       

     According to another embodiment, the vaccine composition comprising at least one polypeptide of  Borrelia burgdorferi  ss, chosen from the sequences SEQ ID NOs: 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85. Preferably, the at least one polypeptide is chosen from SEQ ID NO: 10. 
     In this other embodiment, the vaccine composition may also comprise at least one other polypeptide of  Borrelia burgdorferi  ss,  Borrelia afzelii  or  Borrelia garinii  chosen from SEQ ID NOs: 1, 3 to 7, 11, 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. 
     In addition, in this other embodiment, the vaccine composition may also comprise at least one other polypeptide of  Borrelia burgdorferi, Borrelia afzelii  or  Borrelia garinii  chosen from the groups (c1), (c2) and (c3), 
     said group (c1) comprising SEQ ID NOs: 19 to 28, 
     said group (c2) comprising SEQ ID NOs: 29 to 46 and 73 to 86, 
     said group (c3) comprising SEQ ID NOs: 47 to 72 and 87 to 92, 
     on the condition that, if said at least one polypeptide chosen from SEQ ID NOs: 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85:
         is SEQ ID NO: 19 or 25, said at least one other polypeptide is included in the group (c2) or (c3), or   is SEQ ID NO: 29, 33, 35, 43, 45 or 85, said at least one other polypeptide is included in the group (c1) or (c3), or   is SEQ ID NO: 2, 8 or 9, said at least one other polypeptide is included in the group (c3), or   is SEQ ID NO: 10 or 13, said at least one other polypeptide is included in any of the groups (c1), (c2) and (c3).       

     Advantageously, the composition of the invention may comprise a pharmaceutically acceptable carrier. 
     In the present text, the term “pharmaceutically acceptable carrier” is intended to mean any substance which makes it possible to dilute or transport at least one polypeptide of the vaccine composition according to the invention. Preferably, the pharmaceutically acceptable carrier does not affect the efficacy of the polypeptide. The pharmaceutically acceptable carrier may, for example, be an aqueous solution or an emulsion. 
     When the pharmaceutically acceptable carrier is an aqueous solution, said solution may be, for example, any one of the solutions presented in the document 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 the document Wehrlé P. (Wehrlé P., Pharmacie galénique, Formulation et technologie pharmaceutiques [Galenical pharmacy, Pharmaceutical formulation and technology], 2007 [11]). 
     When the pharmaceutically acceptable carrier is an emulsion, said emulsion may be a water-in-oil, oil-in-water or water-in-oil-in-water emulsion (Wehrlé P. [11]). 
     The vaccine composition according to the invention may comprise an adjuvant. The term “adjuvant” is intended to mean 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 for example be any adjuvant known to those skilled in the art for the administration of polypeptides. 
     For example, the adjuvant may be an adjuvant which induces a humoral response and/or a cellular response. By way of nonlimiting examples, the adjuvant may be chosen from alumina hydroxide, saponin extracts, immune stimulating complexes (also called “ISCOMs”), inulin, Toll receptor (TLR) agonists, Cytosine Phosphate Guanine (CPG) complexes, chitosan or else mycolic acids. It may also be saponin (Roatt et al., 2012 [14]) or alumina hydroxide (Livey et al., 2011 [15]; Wressnigg et al. 2013 [16]). 
     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 pathological condition(s) distinct from Lyme disease. By way of example, the pathological condition(s) distinct from Lyme disease may be chosen from the group comprising leptospirosis, rabies, distemper, parvoviral infection and  Bordetella  infections. 
     According to the invention, the term “used in combination” is intended to mean a use of the vaccine composition according to the invention jointly or simultaneously, concomitantly, or successively, with any known treatment for preventing Lyme disease. The mode of administration may be identical or different according to the molecules coadministered. 
     The term “jointly or simultaneously” is intended to mean the use of the composition according to the invention with any known treatment for preventing Lyme disease in a single composition containing them. 
     The term “concomitantly” is intended to mean the separate use of the vaccine composition according to the invention and of any known treatment for preventing Lyme disease, via identical or different routes of administration during the same administration period. 
     The term “successively” is intended to mean the separate use of the vaccine composition according to the invention and of any known treatment for preventing Lyme disease, via identical or different routes of administration during different administration periods. 
     The term “administration period” is intended to mean the period of 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 one week, two weeks, three 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 may therefore be used for the production of a medicament, in particular a medicament intended for preventing Lyme disease. 
     The vaccine composition for use as a medicament or for use in the prevention of Lyme disease may be intended for any mammal capable of contracting or having contracted Lyme disease. In particular, it may be intended for human beings or for dogs, for horses, for cattle or for 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 appropriate administration form. It may be one of the forms known by those skilled in the art for administering an active molecule which is a polypeptide (Peppas N A, Carr D A, Chemical engineering Science, 64, 4553-4565 (2009) [12]; Morishita M, Peppas N A, Drug Discovery Today, 11, 905-910 (2006) [13]). 
     The vaccine composition according to the present invention may, for example, be intended for administration by injection. 
     Thus, the vaccine composition according to the invention may be packaged in any form known to those skilled in the art for the purpose of being administered by injection. It may, for example, be a bottle or a vial. 
     For example, the injection may be an intramuscular, intradermal or subcutaneous injection. According to one embodiment, the injection is carried out intradermally. According to another advantageous embodiment, the injection is carried out intramuscularly or subcutaneously. 
     The vaccine composition of the present invention may be administered as a medicament, preferably in sufficient amount to prevent Lyme disease, in particular for preventing Lyme disease in dogs. For example, the polypeptides of the vaccine composition according to the invention may be inoculated at doses of between 1 and 500 μg, preferably between 10 and 100 μg (Wressnigg et al., 2013 [16]). 
     The synthesis of the polypeptides that can be used in the vaccine composition according to the present invention may be carried out by any process known to those skilled in the art. It may for example be a synthesis by genetic engineering. 
     When the synthesis of the polypeptides of the vaccine composition according to the invention is carried out by genetic engineering, it is for example possible 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 collect said polypeptide of the vaccine composition according to the invention. The protocol described in F. Cordier-Ochsenbein et al.  J. Mol. Biol.  279, 1177-1185 [17] can, for example, be used. 
     The vaccine composition according to the invention may be produced according to any method well known to those skilled in the art. It may for example be simple mixing of the various constituents of the vaccine composition. The document by Ramamoorthi and Smooker (2009) [18] describes a process for producing a vaccine composition that can be used in the context of the present invention. 
     Thus, the present invention provides effective solutions for the prevention of Lyme disease. 
     Other advantages may become further apparent to those skilled in the art on reading the examples below given by way of nonlimiting illustration. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  represents the PCR quantification of  B. burgdorferi , native strain 297 and of its virulent clone 297c4, in mouse skin, relative to the time after inoculation of the strain. “N Fla/10 4  GAPDH” signifies the 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 mouse skin during early transmission of the bacterium. 
     
    
    
     EXAMPLES 
     Example 1 
     Strategy for Identifying  Borrelia  Vaccine Candidates for Lyme Disease 
     The inventors developed a proteomic approach in order to identify and select effective polypeptides for the prevention of Lyme disease. 
     They selected, by cloning in solid medium (De Martino et al., 2006 [19]), strains of  Borrelia burgdorferi  ss,  Borrelia afzelii  and  Borrelia garinii  that were virulent and nonvirulent on mice. A Gel-LC-MS/MS strategy was used to compare the proteins in each  Borrelia  culture. 
     1.1 Materials and Methods 
     1.1.1 Mice, Bacterial Strains and Culture Conditions 
     C3H/HeN mice three-to-four weeks old were used (Charles River Laboratories, L&#39;Arbresle, France). 
     Three strains of  Borrelia burgdorferi  sensu lato were analyzed: 
       Borrelia burgdorferi  ss, strain 297 (A. Steere, USA), isolated from the cerebrospinal fluid (CSF) of a patient, 
       B. garinii , strain PBi (B. Wilske, Germany) isolated from the CSF of a patient, 
       B. afzelii , strain 163/98 (E. Ruzic-Sabljic, Slovenia) isolated from the CSF of a patient. 
     For each species, the bacteria were cloned on BSK-S. The bacterial clones were then cultured on BSK-H medium (Sigma) and tested on a mouse model according to the protocol described in De Martino et al. [19]. The bacterial clones were selected for their virulence in mice. All the strains were cultured in complete BSK-H medium (Sigma) at 33° C. and used at low passage (&lt;7). The  Borrelia  were counted and the viability was verified by dark-background microscopy. 
     1.1.2 Identification of Proteins, after Digestion with Trypsin and nanoLC-MS/MS 
     For each strain and its clone, the proteins were extracted with a Laemmli buffer [20]. After sonication and centrifugation, the pellet was removed and the protein concentration of the supernatent was determined. The proteins (75 μg) were subjected to a prefractionation step on an SDS-PAGE (12% acrylamide) one-dimensional electrophoresis gel. The resulting lanes were stained with Coomassie blue [21]. Gel bands of 2 mm were systematically manually excised. The digestion of the proteins contained in the gel was carried out as described in Villiers et al. [22], and the (tryptic) peptides obtained were extracted by adding 35 μl of 60% (v/v) of acetonitrile (ACN) and 0.1% (v/v) of HCO 2 H. The nanoLC-MS/MS analysis was carried out using a nanoLC-Chip/MS system (Agilent Technologies, Palo Alto, Calif.) coupled to an amaZon ion trap (Bruker, Bremen, Germany). The chromatographic system was composed of a precolumn (40 nl, 5 μm) and of a column (150 mm×75 μm, 5 μm) comprising the same Zorbax 300SB-C18 stationary phase. The solvent system was composed of 2% of ACN, 0.1% HCO 2 H in water (solvent A) and of 2% water, 0.1% HCO 2 H in ACN (solvent B). 1 μl of peptide extract ( 1/20 of the total volume) was loaded in duplicate onto the precolumn (in the enrichment column) at a charge flow rate of (a flow speed fixed at) 3.75 μl/min with solvent A (at 100% of 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 of the column with 8% of solvent B. The acquisition parameters of the MS and MS/MS spectra are the following: source temperature regulated at 145° C. and gas flow rate at 4 l/min. The voltage applied to the (nanoelectrospray) sprayer needle was regulated at −1900 V. The acquisition of the MS spectra was carried out in positive (ion) mode over a range of masses of 250 to 1500 m/z at a scanning speed of 8100 m/z per s. The maximum number of ions (control of ionic charge) and the maximum accumulation time were respectively fixed at 200 000 and 200 ms, with an average over two scans. The acquisition of the MS/MS spectra was carried out by sequentially selecting the eight most intense (abundant) precursor ions, with a preference for doubly charged ions. The threshold of selection of an ion for the fragmentation (absolute threshold) was fixed at 100 000. The fragmentation was carried out using argon as collision gas. The ions selected were excluded for 0.6 min. The MS/MS spectra were produced over a mass range of from 100 to 2000 m/z. The maximum number of accumulatable ions in MS/MS (control of ionic charge) was fixed at 400 000, with an average over five scans. The complete system was controlled by the Hystar 3.2 software (Bruker). 
     1.1.3 Data Analysis 
     The raw data from the MS and MS/MS spectra (mass data collected during the nanoLC-MS/MS) were processed, converted into “.mgf” files with the DataAnalysis 4.0 software (Bruker) and interpreted using the MASCOT 2.4.3 (Matrix Science, London, United Kingdom) [23] and OMSSA 2.1.7 (Open Mass Spectrometry Search Algorithm, Maryland, USA) [24] algorithms. The searches were carried out without any molecular-weight or isoelectric-point restriction in protein data banks composed respectively of sequences (of proteins) of  Borrelia burgdorferi  ss B31,  Borrelia garinii  PBi and  Borrelia afzelii  PKo downloaded from the nonredundant data bank of the National Centre for Biotechnology Information (NCBInr, Aug. 16, 2012). The known contaminating proteins such as human keratin and trypsin were added to each data bank and linked with reverse copies of all the sequences ( B. burgdorferi  ss B31: 1758 entries;  B. garinii  PBi: 1720 entries;  B. afzelii  PKo: 2157 entries). The  B. burgdorferi  ss B31 and  B. afzelii  PKo data banks were used since the  B. burgdorferi  ss 297 and  B. afzelii  163 strains respectively have not yet been sequenced. Trypsin was chosen as enzyme. The tolerance with respect to the mass of the precursors and of the fragments was fixed at 0.5 Da. A maximum of two mixed cleavages was accepted and a few post-translational modifications were taken into account: carbamidomethylation (C), N-terminal acetylation, oxidation (M). The results of the MASCOT and OMSSA algorithms were independently loaded into the Scaffold software (Proteome Software, Portland, Oreg.). The level of false positives was fixed at 1% with a minimum of one peptide per protein. 
     The number of spectra attributed to each protein within each duplicate was used in order to demonstrate proteins overexpressed in the virulent clones. The beta-binomial test [25] was carried out in R in order to determine the overexpression of the proteins (p&lt;0.05) in each virulent clone compared with the wild-type clone. The test was carried out independently for each of the search engines since the spectral identifications are algorithm-dependent. 
     1.2 Results 
     1.2.1 Reproducibility of the Identification of Bacterial Proteins According to the Infection and the Search Algorithm Used 
     Each sample was analyzed in duplicate. The identification of the proteins present in each repetition was carried out using a combination of the two search engines: Mascot and OMSSA. Thus, the reproducibility of the identifications was evaluated for the two search algorithms. The number of proteins common to the injection duplicates (overlapping) is high (&gt;90% in most cases) and a few proteins were specifically identified in a replicate. This proportion is higher in Mascot compared with OMSSA. These specific identifications could be explained either by the small amount of these proteins, or by the variability due to the data-dependent acquisition (DDA) mode. In order to compare the identifications obtained using the search engines, the identifications of the duplicates were fused. For all the samples, there is a high number (&gt;85%) of (overlapping) proteins identified both by Mascot and by OMSSA. A few proteins are observed specifically by a single algorithm. The combination of the search engines leads to more than 5% of additional identifications. 
     1.2.2 Proteins (Polypeptides) Involved in the Bacterial Transmission 
     For each species, the identifications obtained by Mascot or 
     OMSSA were fused and the protein profiles of the wild-type clones and of the virulent clones were compared by the inventors. More than 800 proteins were identified in each case and a significant intersection (overlap) between the wild-type and virulent clones was observed: around 90% for  B. burgdorferi  ss and  B. garinii  and 80% for  B. afzelii . A higher proportion of proteins detected in the virulent strain compared with the wild-type strain was thus noted for each species (up to 110 for  B. burgdorferi  ss). 
     Moreover, proteins present both in the virulent clone and in the wild-type strain of  Borrelia , but overexpressed in the virulent clone, were detected. Consequently, a strategy based on the number of spectra attributed to each protein (from spectral counting) coupled to an appropriate statistical test (beta-binomial) [25] was used to demonstrate the proteins overexpressed in the virulent clone compared with the wild-type strain (p&lt;0.05). Statistical tests were carried out independently for Mascot and OMSSA since the number of spectra assigned is generally variable between the two search engines [26]. A protein with a p value less than 0.05 both for Mascot and for OMSSA was considered to be overexpressed in order to limit the number of false positives. The number of proteins overexpressed depends on the species under consideration. 31 overexpressed proteins were detected for  B. burgdorferi  ss, 43 for  B. garinii  and 72 for  B. afzelii.    
     The homologous proteins in the three species were determined using the blastp program [27] with an E-value threshold fixed at 10 −3 °. 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). 
     Forty  Borrelia  proteins were thus retained (represented by the 92 sequences of polypeptides of sequence SEQ ID NOs: 1 to 92 in table 1 above) among more than about a thousand proteins identified for the species belonging to this genus.
         Three proteins are common to all the virulent clones and were not detected in wild-type strains: the first, BB0173 (SEQ ID NO: 13) has a von Willebrand type A (VWA) factor domain which is known to be involved in cell adhesion. The second, BB0566 (SEQ ID NO: 10), has a “Sulfate Transporter and Anti-sigma factor antagonist” (STAS) domain. The sigma factors are key elements in the activation of RNA polymerase (RNAP) transcription involved in the regulation of  Borrelia . The third protein, BB0722 (SEQ ID NO: 16), has not yet been described in the literature, but appears to be a bacterial membrane-associated protein.   Other proteins are linked to the RNAP catalytic nucleus: the β unit (rpoC) and the sigma-54 factor (RpoN). For example, BAPKO0873 (SEQ ID NO: 59) contains an ω domain of an RNA polymerase subunit (RpoZ). BB0765 (SEQ ID NO: 27) contains a DNA polymerase domain III (DNAX). These proteins are linked to the RpoN-RpoS pathway which plays an important role in microbial pathogenicity and survival (Radolf et al., 2012 [3]). In  B. burgdorferi , RpoN directly activates the transcription of RpoS which, in turn, controls the expression of the virulence-associated membrane lipoproteins (OspA, OspC, decorin-binding proteins). A nucleotide-associated protein EBfC appears to be an overall regulator of gene expression in  Borrelia . An increase in EBfC levels influences the expression of  B. burgdorferi  genes by about 4.5%, including genes associated with infection. Other proteins involved in DNA replication, recombination and repair (DNA helicase and SBCD exonuclease), or in tRNA processing, have also been identified.   Four proteins identified relate to periplasmic flagellae: FliE, FliP, FlgE and flagellum-specific ATP synthase (Flip. Mobility is crucial for the infectious cycle of  B. burgdorferi  and the periplasmic flagellae are essential for providing the bacteria with sufficient mobility. It has been shown that the inactivation of genes encoding flagellar proteins results in non-mobile bacteria. Another study has shown that the loss of flagellae decreases  B. garinii  infection.   BB0527 (SEQ ID NO: 43) homologous to Baf (Bvg accessory factor) was also identified. This protein has an inhibitory effect on the activity of alkaline phosphatase and therefore directly influences the expression of the P66 outer membrane protein. Among the proteins overexpressed, 5-methylthioadenosine/S-adenosylhomocysteine (SEQ ID NO: 87) was also identified. This protein is an integral part of the methylation cycle. A recent study has shown that the inhibition of this enzyme can attenuate bacterial virulence.   Several proteins identified are involved in carbohydrate metabolism, such as the proteins linked to the phosphotransferase system (PTS), or in the biosynthesis of lipids and metabolism, such as the acyl carrier protein (ACP). Other proteins are hypothetical and do not have a defined function to date.       

     Example 2 
     Study of Skin Inflammation in Mice, after Inoculations of Various Human Pathotypes of  Borrelia burgdorferi  Sensu Stricto 
     The skin constitutes an essential organ in the development of Lyme borreliosis since  Borrelia  is inoculated therein and multiplies therein before disseminating in the organism and reaching the target organs: joints, nervous system and remote skin. 
     Various human clinical isolates of  B. burgdorferi  ss (pathotypes), with various virulence factors of RST type (16S-23S rRNA intergenic spacer type), were selected. The inflammatory responses in the skin in a murine model were compared according to the protocol described hereinafter in order to determine whether the immunity of the skin played a role in the organotropism of the bacterial strains. 
     The mouse in fact constitutes a model of choice for understanding the pathogenicity mechanisms of  B. burgdorferi  sl [28]. 
     2.1 Materials and Methods 
     2.1.1 Mice and Bacterial Strains 
     C3H/HeN mice three-to-four weeks old were used (Charles River Laboratories, L&#39;Arbresle, France). The  Borrelia burgdorferi  sensu stricto strains were isolated from patients suffering from various clinical manifestations: the PBre strain (RST1) from an erythema migrans (EM) (single lesion—Germany), the MR726 strain (RST3) from a multiple erythema migrans (United States), the 1808/03 strain (RST1) from cerebrospinal fluid (Slovenia) and the 297 strain (RST2) also from cerebrospinal fluid (United States). The  Borrelia  clone c297/4 was selected by culturing on solid BSK medium [19]. All the strains were cultured in complete BSK-H medium (Sigma) at 33° C. and used at low passage (&lt;7). The  Borrelia  were counted and the viability was verified by dark-background microscopy. 
     2.1.2 Monitoring of the Infection of the Mouse 
     The mice were infected with 10 3  spirochetes 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 kept under the same conditions as the infected animals. Evaluation of arthritis was carried out every week by measuring the thickness of the two tibiotarsal joints with a metric caliper. Measurements carried out jointly gave an indication of the seriousness of the arthritis. The serology was carried out as described in Kern et al. [29]. 
     At various times after the beginning of the experiment (0 h, 5 h, 24 h, 3 d, 5 d, 7 d, 15 d and 30 d after infection), the mice were killed with an overdose of isoflurane gas. Approximately 1 cm of skin was collected from the site of inoculation and stored in Trizol (registered trademark) (Invitrogen). The ear, the base of the heart, the bladder and the tibiotarsal joints of each mouse were aseptically collected and divided into two parts, for the PCR and the culture of  Borrelia . The organs of the noninfected mice were collected under the same conditions as the positive mice. 
     2.1.3 Detection of  B. Burgdorferi  in the Mouse Organs 
     For the detection of the spirochetes by culture, the organs removed were placed in 6 ml of BSK-H medium containing 30 μg of rifampicin (BioRad). The tubes were kept at 33° C., and the presence of spirochetes was examined every week by dark-background microscopy. 
     For the PCR, the DNA was extracted from the organs of each mouse on a MagNA Pure system (Roche Diagnostics, France), using a MagNA Pure LC large-volume isolation kit after external lysis. The heart, the bladder, the ear and the skin were placed in 500 μl of lysis buffer containing proteinase K. Other samples were treated with external lysis using collagenase A, then proteinase K. All the DNA samples were finally eluted in 100 μl of elution buffer. Ten μL of  Borrelia  DNA were used as a positive control for the detection. The qualitative amplification was carried out as described in Woods et al. [30], by targeting the flagellin gene. 
     2.1.4 Quantification of the Spirochete Load and of the Inflammatory Genes of the Mouse Skin 
     On the site of inoculation, the  B. burgdorferi -specific flagellin gene was quantified on a LightCycler system (Roche Diagnostics, France). The primers used to amplify the fla gene were those described in Kern et al. [29]. 
     To measure the inflammation at the site of inoculation, the total RNA was extracted from 10 mg of mouse skin using the Trizol reagent according to the indications of the manufacturer (Invitrogen). The samples were treated with DNAse (Ambion, USA) and then a first cDNA strand was synthesized from 1 μg of total RNA using the SuperScript II reverse transcriptase (Invitrogen Life Technologies). The gapdh was quantified as an internal standard. The relative expression levels were calculated using an infected animal as calibrator. The amplification and the detection were carried out with an ABI 7000 system with the thermal profile hereinafter: 95° C. for 10 minutes, 50 cycles of 95° C. for 15 s, at 60° C. for 1 min. The primers used for all the genes studied are described in Kern et al. [29]. 
     2.1.5 Comparison of the Protein Profile of the  B. Burgdorferi  Strain 297, of the Wild-Type Strain and of the Virulent Clone c297/4 
     The cultures of  B. burgdorferi  297, of the wild-type strain and of the virulent clone c297/4 were suspended in Laemmli buffer [20]. The protocol presented in examples 1.1.2 and 1.1.3 above was then applied. 
     2.1.6 Dynamics of the Genes of  B. Burgdorferi  297, of the Wild-Type Strain and of the Virulent Clone c297/4, in the Mouse Skin at the Site of Inoculation 
     At various times (0 h, 5 h, 24 h, 3 d, 5 d, 7 d, 15 d and 30 d after infection), skin samples were taken from each mouse at the site of inoculation. The total RNA was purified using the Trizol reagent according to the instructions of the manufacturer. The concentration and the purity of the extracted RNAs were determined by measuring the optical density at A260 and A280. The samples were then treated with gDNAse (Qiagen) in order to remove the contamination with DNA. The total RNAs extracted were subjected to Quantiscript Reverse Transcription (Qiagen) so as to produce the cDNA. The cDNA was used to quantify the ospC and bbk32 genes. For  B. burgdorferi  C297/4, the selected genes corresponding to cell envelope proteins were retained for the RT-PCR. The relative expression levels were calculated using the ΔΔCt method with flagellin as internal standard. The amplification and the detection were carried out with an ABI 7500 system with the thermal profile hereinafter: 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 on day 3 for the relative quantification. The correlation factors were calculated by comparing the cDNA amplification of each point of the time course of the native strain with the cDNA amplification of each point of the hypervirulent clone. Next, the curve obtained for the clone was standardized with these factors so as to obtain a second curve, representative of the wild-type strain and quantitatively comparable to the clone. 
     2.1.7 Statistical Analysis 
     Each experiment was carried out at least three times. For each of the RT-PCRs, at least two extractions were carried out for each mouse in each experiment, with two to three mice for each point. 
     2.2 Results 
     2.2.1 Transmission and Diffusion of the Various Pathotypes of  B. Burgdorferi  ss in Mice 
     All the strains studied showed a similar diffusion tendency.  Borrelia  was detected on day 3 by PCR in the skin, on the site of inoculation, for all the strains. They diffused rapidly into the joint, where they were first detected on day 5 or 7, then to the heart and the bladder on day 5, 7 or 15, and the slowest dissemination occurred in the ear (skin remote from the site of inoculation). The PBre pathotype, isolated from erythema migrans (EM) diffused more slowly to the heart and the bladder, compared with the others. By ELISA, all the mice became positive for the  Borrelia  antigens 15 days after the bacterial inoculation. 
     2.2.2 Quantification of the  Borrelia  Pathotypes and Measurement of the Inflammation at the Site of Inoculation 
     The bacterial load of the skin was measured. All the strains multiplied intensively on day 7, but without any significant difference observed between the strains tested. 
     The inflammatory profile in the skin of the mice was compared for these various strains of  B. burgdorferi  ss. The antimicrobial peptides (AMPs), which are markers of the innate immunity of epithelia, were measured. The PBre strain (EM) induced a significant amount of cathelicidin with a peak on day 3. The MR726 strain (MEM) strongly induced the defensin mBD-3. The wild-type 297 strain (CSF) exhibited an mBD-3 peak at 24 h while the 1808/03 strain (CSF) induced a negligible amount of all of the three AMPs tested. The induction of supplementary pro-inflammatory molecules was then measured: TNF-α, IL-6, IL-22 and the chemokine MCP-1. For each of them, a TNF-α and/or MCP-1 induction peak was observed on day 7. The MR726 strain isolated from an MEM lesion induced the strongest inflammatory profile in the mouse skin with an MCP-1 peak (150 times) on day 7. 
     2.2.3 Specific Analysis of the Inflammation Induced by  B. Burgdorferi  ss 297 Wild-Type Strain and its Hypervirulent Clone 
     The  Borrelia  infection could be initiated with a heterogeneous population of  Borrelia  in the vertebrate host. A  B. burgdorferi  ss 297 clone C297/4 was selected in the laboratory for its rapid diffusion and its neurological manifestations in mice [31]. The virulent clone C297/4 caused an inflammation of the skin with a greater induction of the defensins, MBD-14, and of cathelicidin compared with the wild-type strain. A very strong induction of MCP-1 and of IL-6, approximately 100 times more induction, was observed for the clone C297/4 compared with the wild-type strain. 
     The results of the wild-type 297 strain and of the hypervirulent clone were also compared in the C3H/HeN mice. The hypervirulent clone diffused more rapidly to the joint, whereas the diffusion to the other organs was similar to that of the wild-type strain. The quantification of the bacterial load in the tissues confirmed the intense multiplication occurring in the skin on day 7 regardless of the strain used, but no significant difference was observed between the hypervirulent clone and the wild-type 297 strain. 
     2.2.4 Proteomic Characterization of the Wild-Type 297  B. burgdorferi  ss Strains and of the Hypervirulent Clone 
     The protocol presented in examples 1.2.1 and 1.2.2 above was applied. 
     A total of 887 proteins were identified with 848 c297/4 proteins and 777 proteins in the wild-type strain. An overlap of 738 proteins, which represents 83% of the total number, was observed. 110 proteins are specific for the hypervirulent clone. 
     2.2.5 Comparative Expression of the Proteins Specific for the Wild-Type and Hypervirulent Clones of  B. burgdorferi  297 in the Mouse Skin by RT-PCR 
     The kinetics of expression of OspC and BBK32, two important proteins in the transmission of  Borrelia , were determined for the wild-type and c297/4 strains. The two strains exhibit a first OspC expression peak on day 5, while a BBK32 expression peak was observed on day 7.  Borrelia  surface proteins were then selected among the 110 proteins specific for the hypervirulent clone, and their expression was monitored during the skin inflammation in the C3H/HeN mouse. Three genes are strongly expressed in the two strains, bb0304, bb0213 and bb0347 with an expression peak on day 5 for the hypervirulent clone and on day 7 for the wild-type strain. 
     Example 3 
     Selection of the Vaccine Candidates and Determination of the Immunogenic Effect 
     The various proteins were tested in a C3H/HeN murine model in order to see their expression in the skin, during the transmission of the bacterium. This is because the skin interface appears to play a key role in the selection of certain bacterial populations (Brisson et al., 2011 [32]). The skin of the intradermally infected mice is sampled at 3, 5, 7 and 15 days. After having designed specific primers for each of the proteins, the RT-PCR technique is used to monitor the expression of these proteins in the skin. Those which are the most expressed in the skin are then retained. They are then cloned (Steere et al., 1998 [33]; Ramamoorthi and Smooker, 2009 [18]; Livey et al., 2011 [15]) and expressed in  E. coli . They are inoculated intradermally into the mouse and the antibody titer is measured by ELISA. Indeed, the antibody response appears to be essential for measuring a protective effect during Lyme borreliosis (Embers and Narasimhan, 2013 [34]). Their protective effect is tested by means of a challenge with  Borrelia  inoculated with a syringe, or better still with ticks infected with  Borrelia . The vaccinated and challenged mice are then dissected and their organs cultured or tested by PCR in order to measure the absence of  Borrelia  (Kern et al., 2011 [29]). 
     Five proteins were retained for in vivo tests in mice, namely the three “hypothetical proteins” only detected in the virulent clones and common to the three  Borrelia  species (SEQ ID NOs: 10 (BB0566), 13 (BB0173) and 16 (BB0722) of  Borrelia burgdorferi  ss and the respective corresponding sequences SEQ ID NOs: 11 (BAPKO0596), 14 (BAPKO0175) and 17 (BAPKO0766) of  B. afzelii  and 12 (BG0576), 15 (BG0172) and 18 (BG0744) of  B. garinii ), the RpoN protein (SEQ ID NO: 41 (BB0450) of  Borrelia burgdorferi  ss and the corresponding sequence SEQ ID NO: 42 (BAPKO0472) of  B. afzelii ) and the Gnd protein (SEQ ID NO: 77 (BB0561) of  Borrelia burgdorferi  ss and the corresponding sequence SEQ ID NO: 42 (BAPKO0590) of  B. afzelii ). 
     In parallel, in the skins of infected mice 7 days after intradermal inoculation, all the  Borrelia  proteins expressed in the skins of infected mice were analyzed by a non-targeted proteomic approach. This is because 7 days corresponds to a peak of intense multiplication of the bacteria after intradermal inoculation and therefore probably plays a key role in the initiation of the bacterial infection (Kern et al., 2011 [29]). The strategy consisted in extracting the proteins contained in the infected skins, in prefractionating them by gel electrophoresis and then in identifying them by liquid chromatography coupled to tandem mass spectrometry (Ge-LC-MS/MS strategy). 
     3.1 Materials and Methods 
     3.1.1 Inoculation of Bacteria and Sampling of Infected Skins 
     C3H/HeN mice three-to-four weeks old were purchased from Charles River Laboratories (L&#39;Arbresle, France). 
     The inventors were particularly interested in the  B. burgdorferi  ss 297 strain, isolated from cerebrospinal fluid in the United States (Sterre et al., 1893 [35]). 
     Various clones of  B. burgdorferi  ss were obtained by culturing on a solid BSK medium ([19]). The 297c4 clone was selected for its rate of dissemination in mice and its location in the brain in particular. 
     All the  Borrelia  strains were cultured in BSK-H medium (Sigma) at 33° C. and used at low passage (&lt;7) for the mouse infection. The spirochetes were counted and the viability was verified using a dark-background microscope. The mice were infected with 10 3  spirochetes in 0.1 ml of BSK by intradermal injection in the dorsal thoracic region. 
     At various points after the inoculation (3 d, 5 d, 7 d, 15 d), the mice were killed with isoflurane. An area of 1 cm of mouse skin was collected at the site of inoculation and stored in Trizol (Invitrogen) for the RT-PCR analyses. For the quantification of the  Borrelia  in the skin, the sample is stored dry at −80° C. 
     3.1.2 PCR Quantification of the Skins Infected with  Borrelia    
     At the site of inoculation, the detection of the presence of  B. burgdorferi  ss was carried out by PCR by targeting the flagellin gene on a LightCycler system (Roche Diagnostics, France). The primers used to amplify the fla gene are those previously described ([29]). 
     3.1.3 RT-PCR on the Mouse Skins for Gene Detection 
     At the various points of the time course, the skin samples were taken from each mouse at the site of inoculation. The total RNA was purified using the Trizol reagent according to the instructions of the manufacturer. The concentration and the purity of the RNA extracted were determined by measuring the A260 and A280. The samples were then treated with gDNAse wipeout (Qiagen). The total RNA extracted was synthesized to give cDNA using Quantiscript reverse transcription (Qiagen). The 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 by RT-PCR using the primers described in table 3 below. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Primers used for the RT-PCR 
               
            
           
           
               
               
               
            
               
                   
                 SEQ 
                   
               
               
                   
                 ID 
                   
               
               
                 Proteins 
                 NO: 
                 Sequences 
               
               
                   
               
               
                 BB0566 
                  93 
                 F-AGG CCT GAA GGA GAG CTT GT 
               
               
                   
                  94 
                 R-AAA CCT CAT CGG ATG GAT ACT CAA 
               
               
                   
               
               
                 BB0722 
                  95 
                 F-GCT GAT TTT GCC AGC GAG CTT A 
               
               
                   
                  96 
                 R-TCG GTC CAA ATA CTT CCG TAA CC 
               
               
                   
               
               
                 BB0173 
                  97 
                 F-TCG CCT AGT ATG GGG GCT GTT 
               
               
                   
                  98 
                 R-AGC AGA ACC ATT GCC AAG ATC C 
               
               
                   
               
               
                 RpoN 
                  99 
                 F-AAG TGA AAA CCC CCA AAA ACA AAA A 
               
               
                   
                 100 
                 R-TTG CTC CAC CAA CAG AGC TAA AAA G 
               
               
                   
               
               
                 Gnd 
                 101 
                 F-GGA ATG AAG GCG ATC TTT CAG GG 
               
               
                   
                 102 
                 R-GCT GGC AAA GGA ATC CCA ATT TCAC 
               
               
                   
               
            
           
         
       
     
     The relative expression levels were calculated using the ΔΔCt method with flagellin as internal standard. The amplification and the detection were carried out with an ABI 7500 system with the following thermal profile: 95° C. for 10 min, 50 cycles of 95° C. for 15 s, and 60° C. for 1 min. Each amplification condition was compared on day 3 for the relative quantification. 
     3.1.4 Proteomic Analysis of the Infected Skins 
     The biopsies were selected according to the quantification by PCR. Fragments of approximately 4 mg were cut up and the proteins were extracted in 200 μl of Laemmli buffer and then assayed. The proteins (50 μg) were prefractionated on an SDS-PAGE electrophoresis gel and then the migration lanes were excised and treated 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 MS and MS/MS spectra were acquired with the same parameters and the searches were performed in the same way, except for the data banks. In the present case, the searches were performed in data banks composed of  B. burgdorferi  ss B31 and mouse sequences, downloaded from the NCBInr and UniProtKB-SwissProt data bank, respectively ( B. burgdorferi  B31: Aug. 16, 2012; mouse: Apr. 19, 2013). 
     3.2 Results 
     3.2.1  Borrelia  Multiplication Peak on Day 7: 
     The quantification of the  Borrelia  in the skin at various points after intradermal inoculation reveals an intense multiplication of the bacteria on day 7, this being regardless of the  Borrelia  strain tested ( FIG. 1 ). 
     3.2.2 Kinetics of Expression of Certain Genes in the Skin 
     The expression profile, by RT-PCR, of the BB0566 protein (SEQ ID NO: 10), which is a protein common to the three species of  Borrelia: B. burgdorferi  ss,  B. afzelii  and  B. garinii , in the mouse skin during the early transmission of the bacterium is represented in  FIG. 2 . 
     These results show that the protein of sequence SEQ ID NO: 10 is strongly overexpressed in the mouse skin from the fifth day after inoculation for the two strains 297 and 297c4. 
     3.2.3 Proteomic Analysis of the Skins of Mice Infected on Day Seven after Intradermal Inoculation 
     On average, 1350 mouse proteins were identified in the skin biopsies from infected mice. Among the  Borrelia  proteins detected, the RpoN and Gnd proteins were identified, thereby confirming the expression of these proteins in the skin seven days after inoculation and their potential role during the early transmission of the bacterium. 
     Example 4 
     Vaccine Trial 
     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 μg. According to the vaccination protocol in dogs, ideally, two administrations will be carried out, 2 to 4 weeks apart, followed by an annual booster. The adjuvant is chosen according to its ability to stimulate the humoral response and/or the cellular response. Those skilled in the art know how to determine which adjuvant to choose in order to efficiently stimulate the humoral response and/or the cellular response. The vaccine is administered intradermally, subcutaneously or intramuscularly, preferably intramuscularly or subcutaneously. 
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