EP3033105A2

EP3033105A2 - Lyme disease vaccines

Info

Publication number: EP3033105A2
Application number: EP14786957.2A
Authority: EP
Inventors: Benoît JAULHAC; Nathalie BOULANGER; Laurence Sabatier; Gilles SCHNELL
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Strasbourg
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Strasbourg
Priority date: 2013-08-14
Filing date: 2014-08-12
Publication date: 2016-06-22
Also published as: FR3009683A1; US20160220653A1; WO2015022470A2; WO2015022470A3

Abstract

The present invention relates to Lyme disease vaccines, in particular to vaccines including one or more isolated polypeptides of Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii.

Description

VACCINES AGAINST LYME DISEASE

Technical area

The present invention relates to vaccines against Lyme disease, in particular vaccines comprising one or more polypeptides isolated from Borrelia bu rdorferi ss, Borrelia afzelii or Borrelia garinii.

The present invention has applications in the veterinary and medical fields. In the description below, references in brackets ([]) refer to the list of references at the end of the examples.

State of the art

Lyme borreliosis, also known as Lyme disease, is a vector-borne disease transmitted by a hard tick of the genus Ixodes. It occurs mainly in the Northern Hemisphere where it is the most common vector-borne disease. Recent data also suggest that its range also extends into the southern hemisphere with human cases in Australia (Mayne et al., 201 1 [1]) and ticks infected with Borrelia identified in South America ( Barbieri et al., 2013 [2]).

The bacterium responsible for borreliosis is a spirochete belonging to the group Borrelia burgdorferi sensu lato with about 20 identified species.

Lyme borreliosis usually develops in wildlife in a wide range of vertebrate hosts and is manifested accidentally in humans first by skin inflammation, erythema migrans, then by a wide variety of clinical manifestations: articular, cardiac, neurological and cutaneous (Radolf et al., 2012

[3]; Stanek et al., 2012 [4]). The skin thus 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 infection, many patients develop borreliosis due to absence or non-observation of erythema migrans, or due to inadequate or inappropriate treatment. late. A vaccine approach appears to be more promising for preventing or treating Lyme disease, especially in animals where the early skin stage can not be observed due to peeling.

There are currently several vaccines on the market to prevent canine borreliosis. In the United States, they are only directed against a single species, Borrelia burgdorferi sensu stricto. In addition, based on the concept of "transmission blocking vaccine", a vaccine was marketed in the United States for humans, the LYMErix (trademark), but its marketing was stopped in 2002 following side effects in some 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) marketed by Intervet and Recombitek (trademark) marketed by Merial and have shown some effectiveness but only against the species B. burgdorferi ss (Lafleur et al., 2009 [7]), because the population of Borrelia transmitted by ticks is in Europe very heterogeneous, and vaccines currently on the market are not effective against Borrelia's other major viral species in Europe A third vaccine is marketed using a bacterial lysate of B. burgdorferi ss (Fort Dodge) In Europe, only two companies (Merial and Bioveta) market a vaccine also based on lysates of Borrelia, B. burgdorferi ss for Merial and B. afzelii and B. garinii for Bioveta.These vaccines, generally three injections are necessary to obtain sufficient protection, generally controlled by the measurement of antibodies by ELISA (Topfer and Straubinger, 2007 [8]). However, the use of bacterial lysate as the vaccine base is unsatisfactory for mass production.

Thus, existing vaccines use either bacterial lysates whose mass production is difficult to achieve, or the recombinant protein OspA, poorly immunogenic and poorly expressed at the beginning of the infection in humans, or OspC whose proof of concept on a vaccination plan is not proven.

There are therefore real needs to develop new vaccines overcoming these defects, disadvantages and obstacles of the prior art, in particular vaccines which are highly immunogenic and effective on various populations of Borrelia, and which can be mass produced at low cost.

Presentation of the invention

The present invention precisely meets the aforementioned 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 to identify and select effective polypeptides for the prevention of Lyme disease. This approach was carried out on the basis of three species of Borrelia that the inventors have determined to be the most involved in human and animal pathology, in particular in dogs, namely Borrelia burgdorferi ss, Borrelia afzelii and

Borrelia garinii.

Thus, the subject of the present invention is in particular a vaccine composition comprising at least one Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide chosen from the sequences described below. In particular, the sequences SEQ ID NO: 1 to 92 shown in Table 1 below, in which the sequence numbers of the appended sequence listing, the names of the corresponding polypeptides and the names of the corresponding loci in the genome of the invention are described. Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii.

In other words, Table 1 describes the isolated polypeptides consisting of a sequence chosen from SEQ ID NO: 1 to 92 that may be used in the vaccine composition of the invention.

TABLE 1 Sequences of the Polypeptides Which Can Be Used in a Vaccine Composition According to the Invention

In the present invention, unless otherwise indicated, "Borrelia burgdorferi" means "Borrelia burgdorferi ss" or "Borrelia burgdorferi sensu stricto" as opposed to "Borrelia burgdorferi sensu lato" which covers about 20 different species.

In addition, unless otherwise indicated, the sequence-determining letters of the polypeptides described herein correspond to the one-letter abbreviation proposed by Leder (Leder et al., Introduction to Molecular Medicine, Ed Scientific American, 1994 [9]).

Thus, the subject of the present invention is in particular a vaccine composition comprising at least one Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide chosen from the sequences SEQ ID NO: 1 to 92. Preferably, the at least one polypeptide is chosen. among the sequences SEQ ID NO: 10 to 92, preferably 10 to 18, preferably 10 to 15.

Any polypeptide of sequence SEQ ID NO: 1 to 92, or any combination of at least two of the polypeptides of sequence SEQ ID NO: 1 to 92, may be used in the vaccine composition according to the invention.

For example, the combination of polypeptides may comprise 2 different polypeptides of sequence SEQ ID NO: 1 to 92, for example 3, 4, 5, 6, 7, 8, 9 or even more than 9 different polypeptides of sequence SEQ ID NO: 1 at 92. A combination of the different polypeptides of sequence SEQ ID NO: 1 to 92 may in fact make it possible to increase the therapeutic and / or prophylactic effect of the vaccine composition according to the invention. For example, the vaccine composition according to the invention may comprise a combination of two polypeptides as shown in Table 2 below. Table 2: Combination of polypeptide in the vaccine composition according to the invention

polypeptide

Possible combination with the polypeptides of sequence SEQ NO: sequence following

SEQ ID NO:

At least one of: 2 to 92,

1

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 and 3 to 92,

2

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1, 2 and 4 to 92,

3

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 3 and 5 to 92,

4

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 4 and 6 to 92,

5

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 5 and 7 to 92,

6

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 6 and 8 to 92,

7

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 7 and 9 to 92,

8

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 8 and 10 to 92,

9

preferably at least one of: 10 to 18, 47 to 72 and 87 to 92

At least one of: 1 to 9 and 1 to 92,

10

preferably at least one of: 13 to 18

At least one of: 1 to 10 and 12 to 92,

1 1

preferably at least one of: 13 to 18

At least one of: 1 to 1 1 and 13 to 92,

12

preferably at least one of: 13 to 18

At least one of: 1 to 12 and 14 to 92,

13

preferably at least one of: 10 to 12 and 16 to 18

At least one of: 1 to 13 and 15 to 92,

14

preferably at least one of: 10 to 12 and 16 to 18

At least one of: 1 to 14 and 16 to 92,

15

preferably at least one of: 10 to 12 and 16 to 18

At least one of: 1 to 15 and 17 to 92,

16

preferably at least one of: 10 to 15

At least one of: 1 to 16 and 18 to 92,

17

preferably at least one of: 10 to 15

At least one of: 1 to 17 and 19 to 92,

18

preferably at least one of: 10 to 15

At least one of: 1 to 18 and 20 to 92,

19

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 19 and 21 to 92,

20

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 20 and 22 to 92,

21

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 21 and 23 to 92,

22

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 22 and 24 to 92,

23

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 23 and 25 to 92,

24

preferably at least one of: 10 to 18 and 29 to 92 At least one of: 1 to 24 and 26 to 92,

25

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 25 and 27 to 92,

26

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 26 and 28 to 92,

27

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 27 and 29 to 92,

28

preferably at least one of: 10 to 18 and 29 to 92

At least one of: 1 to 28 and 30 to 92,

29

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 29 and 31 to 92,

30

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 30 and 32 to 92,

31

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 31 and 33 to 92,

32

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 32 and 34 to 92,

33

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 33 and 35 to 92,

34

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 34 and 36 to 92,

35

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 35 and 37 to 92,

36

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 36 and 38 to 92,

37

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 37 and 39 to 92,

38

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 38 and 40 to 92,

39

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 39 and 41 to 92,

40

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 40 and 42 to 92,

41

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 41 and 43 to 92,

42

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 42 and 44 to 92,

43

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 43 and 45 to 92,

44

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 44 and 46 to 92,

45

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 45 and 47 to 92,

46

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 46 and 48 to 92,

47

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 47 and 49 to 92,

48

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 48 and 50 to 92,

49

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 49 and 51 to 92,

50

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 50 and 52 to 92,

51

preferably at least one of: 1 to 46 and 73 to 86 At least one of: 1 to 51 and 53 to 92,

52

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 52 and 54 to 92,

53

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 53 and 55 to 92,

54

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 54 and 56 to 92,

55

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 55 and 57 to 92,

56

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 56 and 58 to 92,

57

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 57 and 59 to 92,

58

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 58 and 60 to 92,

59

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 59 and 61 to 92,

60

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 60 and 62 to 92,

61

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 61 and 63 to 92,

62

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 62 and 64 to 92,

63

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 63 and 65 to 92,

64

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 64 and 66 to 92,

65

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 65 and 67 to 92,

66

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 66 and 68 to 92,

67

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 67 and 69 to 92,

68

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 68 and 70 to 92,

69

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 69 and 71 to 92,

70

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 70 and 72 to 92,

71

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 71 and 73 to 92,

72

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 72 and 74 to 92,

73

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 73 and 75 to 92,

74

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 74 and 76 to 92,

75

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 75 and 77 to 92,

76

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 76 and 78 to 92,

77

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 77 and 79 to 92,

78

preferably at least one of: 10 to 28,47 to 72 and 87 to 92 At least one of: 1 to 78 and 80 to 92,

79

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 79 and 81 to 92,

80

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 80 and 82 to 92,

81

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 81 and 83 to 92,

82

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 82 and 84 to 92,

83

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 83 and 85 to 92,

84

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 84 and 86 to 92,

85

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 85 and 87 to 92,

86

preferably at least one of: 10 to 28,47 to 72 and 87 to 92

At least one of: 1 to 86 and 88 to 92,

87

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 87 and 89 to 92,

88

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 88 and 90 to 92,

89

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 89, 91 and 92,

90

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 90 and 92,

91

preferably at least one of: 1 to 46 and 73 to 86

At least one of: 1 to 91,

92

preferably at least one of: 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 from those of the combination, or 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 NO: 1 to 92. One skilled in the art clearly understands that sequences having a homology or an identity with these sequences can also be used. to be used, equivalently, in the vaccine composition according to the invention, since they have the same effect as the polypeptides of sequence SEQ ID NO: 1 to 92, namely an immunogenic effect, useful in the prevention of Lyme disease. Those skilled in the art are able to identify homologous sequences from the sequences of the polypeptides that can be used in the vaccine composition according to the invention. For example, a sequence used may have a homology or identity greater than 80% with a sequence described in Table 1, for example an identity or homology greater than 85%, or 90%, or 95%, or 99% with a sequence described in Table 1. Various methods, well known to those skilled in the art, can be used to determine the homology between several sequences. This may be, for example, the Basic Local Alignment Search Tool (BLAST) method described in Altschul, SF et al., J. Mol. Biol. 1990 [27].

In Table 1 above, the polypeptide sequences appearing on the same line represent the same protein whose name is indicated in the left-hand column. "N / a" means that the name of the protein in question has not been identified or is not yet known.

Although the polypeptides on the same line represent the same protein, their amino acid sequences are not strictly identical. This may be due to possible mutations that have occurred distinctly in different species of the genus Borrelia. By way of example, the sequences SEQ ID NO: 10, 11 and 12, isolated from the species Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii respectively, represent the same protein.

Polypeptides that can be used in the vaccine composition according to the invention may be specific to a given Borrelia species, or common to two or three Borrelia species selected from Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii. For example :

the polypeptides of sequence SEQ ID NO: 1 to 9 are proteins specific to the virulent clone of Borrelia burgdorferi ss;

the polypeptides of sequence SEQ ID NO: 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 NO: 19 to 28 are proteins common to the virulent clones of Borrelia burgdorferi ss and Borrelia garinii;

the polypeptides of sequence SEQ ID NO: 29 to 46 are proteins common to the virulent clones of Borrelia burgdorferi ss and

Borrelia afzelii;

the polypeptides of sequence SEQ ID NO: 47 to 72 are proteins common to the two virulent clones of Borrelia afzelii and Borrelia garinii;

the polypeptides of sequence SEQ ID NO: 73 to 86 are proteins common to Borrelia burgdorferi ss and Borrelia afzelii; and

the polypeptides of sequence SEQ ID NO: 87 to 92 are proteins common to Borrelia afzelii and Borrelia garinii.

The polypeptides of SEQ ID NO: 1 to 9, 13 to 18, 29 to 32, 37, 38, 51, 52, 57, 58, 71, 72 and 87 to 92 are membrane proteins of Borrelia.

Advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NO: 1 to 92, the polypeptides are polypeptides of different sequences. Advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NO: 1 to 92, the polypeptides represent different proteins.

Advantageously, when the composition comprises a combination of polypeptides of sequence SEQ ID NO: 1 to 92, the polypeptides may be proteins whose combination makes it possible to obtain immunization simultaneously against two species or the three species Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii. In other words, advantageously, the composition according to the invention comprises a protein common to the three aforementioned species or a mixture of several proteins, for example, 2, 3 or 4 proteins, even plus, covering these three species. This embodiment makes it possible to provide universal vaccine compositions with regard to Borrelia populations. According to a 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 NO: 2, 7 to 15, 19, 20, 25, 26, 29, 30, 33 to 36, 41 to 50, 53, 54, 57 to 72 and 85 to 92. Preferably, the at least one polypeptide is chosen from SEQ ID NO: 10 to 15.

In this embodiment, the vaccine composition may further comprise at least one other Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide selected from SEQ ID NO: 1, 3 to 6, 16 to 18, 21 to 24, 27, 28, 31, 32, 37-40, 51, 52, 55, 56 and 73-84.

In addition, in this embodiment, the vaccine composition may further comprise at least one other Borrelia burgdorferi polypeptide, Borrelia afzelii or Borrelia garinii selected from groups (c1), (c2) and (c3),

said group (d) comprising SEQ ID NO: 19 to 28,

said group (c2) comprising SEQ ID NOS: 29-46 and 73-86, said group (c3) comprising SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID

NO: 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 groups (c1), (c2) or (c3), said at least one other polypeptide is included in a group (c1), (c2) or (c3) different, or

is SEQ ID NO: 2, 7, 8 or 9, said at least one other polypeptide is included in 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 Borrelia burgdorferi ss polypeptide, chosen from the sequences SEQ ID NO: 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85. Preferably, the at least one polypeptide is selected from SEQ ID NO: 10.

In this further embodiment, the vaccine composition may further comprise at least one other Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide selected from SEQ ID NO: 1, 3 to 7, 1 1, 12, 14 to 18, 20 to 24, 26 to 28, 30 to 32, 34, 36 to 42, 44, 46 to 84 and 86 to 92.

In addition, in this other embodiment, the vaccine composition may further comprise at least one other Borrelia burgdorferi polypeptide, Borrelia afzelii or Borrelia garinii selected from groups (c1), (c2) and (c3),

said group (d) comprising SEQ ID NO: 19 to 28,

said group (c2) comprising SEQ ID NOS: 29-46 and 73-86, said group (c3) comprising SEQ ID NOS: 47-72 and 87-92, provided that said at least one polypeptide selected from SEQ ID NO : 10, 2, 8, 9, 13, 19, 25, 29, 33, 35, 43, 45 and 85:

- is SEQ ID NO: 19 and 25, said at least one other polypeptide is included in group (c2) or (c3), or

is SEQ ID NO: 29, 33, 35, 43, 45 or 85, said at least one other polypeptide is included in group (c1) or (c3), or

is SEQ ID NO: 2, 8 or 9, said at least one other polypeptide is included in group (c3), or

is at least one other polypeptide is included in any one of the groups (c1), (c2) and (c3). Advantageously, the composition of the invention may comprise a pharmaceutically acceptable carrier

The term "pharmaceutically acceptable carrier" is understood herein to mean any substance that makes it possible to dilute or transport at least one polypeptide of the vaccine composition according to the invention. Preferably, the pharmaceutically acceptable carrier does not affect the effectiveness of the polypeptide. The pharmaceutically acceptable carrier may, for example, be an aqueous solution or an emulsion.

When the pharmaceutically acceptable carrier is an aqueous solution, it may be, for example, any of the solutions presented in Heitz et al., 2009 (Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Heitz F et al. , Br J Pharmacol 2009 May; 157 (2): 195-206 [10]) or in Wehrle P. (Wehrle P., Galenic Pharmacy, Formulation and Pharmaceutical Technology, 2007 [11]).

When the pharmaceutically acceptable carrier is an emulsion, it can be a water-in-oil, oil-in-water, water-in-oil emulsion (Wehrle P. [11]). The vaccine composition according to the invention may comprise an adjuvant. The term "adjuvant" means any substance capable of facilitating and amplifying the immune response to the at least one polypeptide of the vaccine composition according to the invention. It may be for example any adjuvant known to those skilled in the art for the administration of polypeptides.

For example, the adjuvant may be an adjuvant inducing a humoral response and / or a cellular response. By way of nonlimiting examples, the adjuvant may be chosen from alumina hydroxide, saponin extracts, immune stimulation complexes (also called "ISCOM"), inulin, receptor agonists of the type Toll

(TLR), Cytosine Phosphate Guanine (CPG) complexes, Chitosan or still mycolic acids. It may also be saponin (Roatt et al., 2012 [14]) or alumina hydroxide (Livey et al., 201 1

[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 pathology (s) distinct from Lyme disease. For example, the distinct pathology (s) of Lyme disease can be selected from the group consisting of leptospirosis, rabies, distemper, parvovirus and Bordetella infections.

According to the invention, the term "used in combination" means a use of the vaccine composition according to the invention jointly or concomitantly, concomitantly or sequentially, with any known treatment for preventing Lyme disease. The mode of administration may be identical or different depending on the co-administered molecules.

"Joint or simultaneous" means the use of the composition according to the invention with any known treatment for preventing Lyme disease in a single composition containing them.

The term "concomitant" means the separate use of the vaccine composition according to the invention and any known treatment for preventing Lyme disease, by the same or different administration routes during the same period of administration.

"Successive" means the separate use of the vaccine composition according to the invention and any known treatment for preventing Lyme disease, by the same or different administration routes during different periods of administration.

The term "administration period" refers to the time during which a treatment is administered. It may, for example, be several days, for example two days, three days, four days, etc., for example one or more weeks, for example a week, two weeks, three weeks. weeks, etc., for example one or more months, for example one month, two months, three months, etc., for example one or more years, for example one year, two years, three years, etc. The present invention also relates to a vaccine composition according to the invention for use as a medicament.

The present invention also relates to a vaccine composition according to the invention, for use in the prevention of Lyme disease.

The vaccine composition according to the invention can therefore be used for the manufacture of a medicament, in particular a medicament intended to prevent Lyme disease.

The vaccine composition for use as a medicament or for use in the prevention of Lyme disease may be for any mammal susceptible to contracting or having contracted Lyme disease. In particular, it may be for humans or dogs, horses, cattle or other ruminants. Preferably, the vaccine composition according to the invention is intended for the prevention of Lyme disease in dogs.

The vaccine composition according to the invention, used as a medicament, may be in any form of appropriate administration. It may be one of the forms known to those skilled in the art to administer an active molecule which is a polypeptide (Peppas NA, Carr DA, Chemical Engineering Science, 64, 4553-4565 (2009) [12]; Morishita M

Peppas NA, Drug Discovery Today, 11, 905-910 (2006) [13]).

The vaccine composition according to the present invention may, for example, be for administration by injection.

Thus, the vaccine composition according to the invention can be packaged in any form known to those skilled in the art in order to be administered by injection. It can be for example a bottle or a bulb.

For example, the injection may be an intramuscular, intradermal or subcutaneous injection. According to one embodiment, the injection is performed 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 an amount sufficient to prevent Lyme disease, particularly to prevent that in dogs. For example, the polypeptides of the vaccine composition according to the invention can be inoculated at doses of between 1 and 500 g, preferably between 10 and 100 g (Wressnigg et al., 2013 [16]). The synthesis of the polypeptides that can be used in the vaccine composition according to the present invention can be carried out by any method known to those skilled in the art. It can be for example a synthesis by genetic engineering.

When the synthesis of the polypeptides of the vaccine composition according to the invention is carried out by genetic engineering, it is possible, for example, to construct a large polypeptide comprising the polypeptide of the vaccine composition of the present invention and to digest it with restriction enzymes in order to recovering said polypeptide from the vaccine composition according to the invention. For example, the protocol described in F. Cordier-Ochsenbein et al. J. Mol. Biol. 279.1 177-1 185 [17].

The vaccine composition according to the invention can be produced according to any method well known to those skilled in the art. It may be for example a simple mixture of the various components of the vaccine composition. The document by Ramamoorthi and Smooker (2009) [18] describes a method of manufacturing a vaccine composition that can be used in the context of the present invention. Thus, the present invention provides effective solutions for the prevention of Lyme disease.

Other advantages may still appear to those skilled in the art on reading the examples below given for illustrative and non-limiting.

Brief description of the figures

FIG. 1 represents the PCR quantification of B. burgdorferi, native strain 297 and its virulent clone 297c4, in the skin of mice, with respect to the time after inoculation of the strain. "N Fia / 10 ⁴ GAPDH" means number of flagellin per 10 ⁴ of Glyceraldehyde 3-phosphate dehydrogenase.

FIG. 2 represents an expression profile by RT-PCR of a protein common to the three species of Borrelia: B. burgdorferi ss, B. afzelii and B. garinii, namely BB0566 (SEQ ID NO: 10) in the Mouse skin during the early transmission of the bacteria.

EXAMPLES

Example 1: Strategy for identifying Borrelia vaccine candidates for Lyme disease

The inventors have developed a proteomic approach to identify and select effective polypeptides for the prevention of Lyme disease.

They selected by cloning in solid medium (De Martino et al., 2006 [19]), strains of Borrelia burgdorferi ss, Borrelia afzelii and Borrelia garinii virulent and non-virulent in mice. A Gel-LC-MS / MS strategy was used to compare the proteins in each Borrelia culture. 1.1 Material 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'ArbresIe, 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]. Bacterial clones were selected for their virulence in mice. All strains were grown in complete BSK-H (Sigma) at 33 ° C and used at low passage (<7). Borrelia were counted and viability was verified by dark field microscopy.

1 .1 .2 Identification of proteins, after trypsin digestion 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 supernatant was determined. Proteins (75 g) underwent a one-dimensional gel electrophoresis prefractionation step (electrophoresis) SDS-PAGE

(12% acrylamide). The resulting tracks were stained with Coomassie blue [21]. 2 mm gel strips were excised manually and systematically. Digestion of the proteins contained in the gel was carried out as described in Villiers et al.

[22], and the (trypsic) peptides obtained were extracted by the addition of

35 μl of 60% (v / v) acetonitrile (ACN) and 0.1% (v / v) HCO ₂ H. NanoLC-MS / MS analysis was performed using a nanoLC-Chip / MS system (Agilent Technologies, Palo Alto, CA) coupled to an amaZon ion trap (Bruker, Bremen, Germany). The chromatographic system consisted of a pre-column (40 nL, 5 μιτι) and a column (150 mm x 75 μιτι, 5 μιτι) comprising the same Zorbax stationary phase 300SB-C18. The solvent system consisted of 2% ACN, 0.1% HCO ₂ H in water (solvent A) and 2% water, 0.1% HCO ₂ H in ACN (solvent B). 1 μl of peptide extract (1/20 of the total volume) was loaded in duplicate on the pre-column (in the enrichment column) at a loading rate of (a flow rate set at) 3.75 μΙ / min with solvent A (100% solvent A). The elution was carried out at a flow rate of 300 nl / min by application of a linear gradient of 8-40% of solvent B for 30 minutes followed by a step of 4 min at 70% of solvent B before reconditioning. the 8% solvent column B. The acquisition parameters of the MS and MS / MS spectra are as follows: source temperature set at 145 ° C. and gas flow rate at 4 L / min. The voltage applied to the needle of the sprayer (nanoelectro-fogging) was set at -1900 V. The acquisition of the MS spectra was performed in positive (ions) mode over a mass range of 250 to 1500 m / z at a scanning speed of 8100 m / z per sec. The maximum number of ions (ionic charge control) and the maximum accumulation time were respectively set at 200,000 and 200 ms, with an average of two scans. Acquisition of the MS / MS spectra was performed by sequentially selecting the 8 most intense (abundant) precursor ions, with a preference for the dicharged ions. The ion selection threshold for fragmentation (absolute threshold) was set at 100,000. Fragmentation was performed using argon as the collision gas. The selected ions were excluded for 0.6 min. The MS / MS spectra were carried out over a mass range ranging from 100 to 2000 m / z. The maximum number of ions accumulated in MS / MS (control ionic charge) was set at 400,000, with an average of 5 scans. The complete system was driven by the Hystar 3.2 (Bruker) software.

1 .1 .3 Data Analysis

Raw data from MS and MS / MS spectra (mass data collected during nanoLC-MS / MS) were processed, transformed into ".mgf" files with DataAnalysis 4.0 software (Bruker) and interpreted using MASCOT 2.4 algorithms. 3 (Matrix Science, London, United Kingdom) [23] and OMSSA 2.1.7 (Open Mass Spectrometry Search Algorithm, Maryland, USA). [24] The searches were carried out without any restriction of molecular weight or isoelectric point in protein databases consisting of (protein) sequences of Borrelia burgdorferi ss B31, Borrelia garinii PBi and Borrelia afzelii PKo respectively downloaded from the non-redundant database of the National Biotechnology Information Center (NCBInr, 16 August 2012) .The known contaminating proteins such as human keratin and trypsin, were added to each data bank and linked with inverted 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 databases were used because the B. burgdorferi ss 297 and B. afzelii 163 strains respectively have not yet been sequenced. Trypsin was chosen as the enzyme. Mass tolerance of precursors and fragments was set at 0.5 Da. A maximum of 2 missed cleavages was accepted and some pos-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, OR). The false positive rate was set at 1% with a minimum of one peptide per protein.

The number of spectra attributed to each protein in each duplicate was used to detect expressed in virulent clones. The beta-binomial assay [25] was performed at R to determine protein overexpression (p <0.05) in each virulent clone relative to the wild-type strain. The test was performed independently for each of the search engines because the spectral identifications are dependent on the algorithms.

1.2 Results

1 .2.1 Reproducibility of the identification of bacterial proteins according to the injection and the search algorithm used

Each sample was analyzed in duplicate. The identification of the proteins present in each repetition was performed 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 duplicate injection (overlap) is high (> 90% in most cases) and some proteins have specifically been identified in a replicate. This proportion is higher in Mascot compared to OMSSA. These specific identifications could be explained either by the small quantity of these proteins or by the variability due to the data dependent acquisition mode (DDA). In order to compare the identifications obtained using the search engines, we have merged the identifications of the duplicates. For all samples, there is a high number (> 85%) of identified proteins (overlap) by both Mascot and OMSSA. Some proteins are specifically observed by a single algorithm. The combination of search engines leads to more than 5% additional identifications.

1 .2.2 Proteins (Polvpeptides) involved in bacterial transmission

For each species, the identifications obtained by Mascot or OMSSA were fused and the protein profiles of the wild clones and virulent clones were compared by the inventors. More than 800 Proteins were identified in each case and an important recovery (overlap) between wild and virulent clones was observed: nearly 90% for B. burgdorferi ss and B. garinii and 80% for B. afzelii. Thus, for each species, a higher proportion of proteins detected in the virulent clones relative to the wild strain (up to 1 10 for B. burgdorferi ss) was found.

Moreover, proteins present in both the virulent clone and the wild Borrelia strain but overexpressed in the virulent clone, were detected. Therefore, a strategy based on the number of spectra attributed to each protein (spectral count) coupled with an adequate statistical test (beta-binomial) [25] was used to highlight the overexpressed proteins in the virulent clone. compared to the wild strain (p <0.05). Statistical tests were performed independently for Mascot and OMSSA because the number of spectra assigned is generally variable between the two search engines [26]. A protein with a p value below 0.05 for both Mascot and OMSSA was considered over-expressed to limit the number of false positives. The number of overexpressed proteins depends on the species considered. 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 set at ^10-30 . Three proteins are thus common to the three Borrelia species analyzed and 27 proteins are common to at least two of the three species (see Table 1 above).

Forty Borrelia proteins were thus retained (represented by the 92 polypeptide sequences of sequence SEQ ID NO: 1 to 92 in Table 1 above) among more than a thousand proteins identified for the species belonging to this genus.

- Three proteins are common to all virulent clones and have not been detected in wild strains: the first, BB0173 (SEQ ID NO: 13) has a von Willebrand factor A (VWA) domain that is known to be involved in cell adhesion. The second, BB0566 (SEQ ID NO: 10), has a "Sulfate Transporter and Anti-sigma factor antagonist" type domain (STAS). Sigma factors are key elements in the activation of RNA polymerase (ARNP) transcription involved in Borrelia regulation. The third protein, BB0722 (SEQ ID NO: 16), has not yet been described in the literature, but appears to be a membrane-associated protein of the bacterium.

- Other proteins are linked to the catalytic nucleus RNAP: the β unit

(rpoC) and the sigma-54 factor (RpoN). For example, BAPKO0873 (SEQ ID NO: 59) contains a domain ω of an RNA polymerase (RpoZ) subunit. BB0765 (SEQ ID NO: 27) contains a domain III of the DNA polymerase (DNAX). These proteins are linked to the RpoN-RpoS pathway, which plays an important role in pathogenicity and microbial survival (Radolf et al., 2012 [3]). In B. burgdorferi, RpoN directly activates the transcription of RpoS which, in turn, controls the expression of membrane-associated lipoproteins associated with virulence (OspA, OspC, decorin-binding proteins). A protein associated with an EBfC nucleotide appears as a global regulator of gene expression in Borrelia. The increase in EBfC levels influences the expression of B. burgdorferi genes by 4.5%, including genes associated with infection. Other proteins involved in DNA replication, recombination and repair (DNA helicase and SBCD exonuclease), or in tRNA processing have also been identified.

- Four identified proteins are related to the periplasmic flagella: FliE, FliP, FlgE and flagellum-specific ATP synthase (FIN). Mobility is crucial for the infectious cycle of B. burgdorferi and periplasmic flagella is essential to ensure adequate mobility to bacteria. Inactivation of genes coding for flagella proteins has been shown to lead to non-motile bacteria. A Another study showed that flagella loss decreases B. garinii infection.

- BB0527 (SEQ ID NO: 43) homologous to Baf (accessory factor Bvg) has also been identified. This protein has an inhibitory effect on the activity of alkaline phosphatase and thus directly influences the expression of the outer membrane protein P66. Of the overexpressed proteins, 5-methylthioadenosine / S-adenosylhomocysteine (SEQ ID NO: 87) has also been identified. This protein is an integral part of the methylation cycle. A recent study has shown that inhibition of this enzyme can attenuate bacterial virulence.

- Several identified proteins are involved in carbohydrate metabolism such as phosphotransferase-related protein (PTS), or in lipid biosynthesis and metabolism, such as the acyl-bearing protein (ACP). Other proteins are hypothetical and have no definite function to date.

Example 2 Study of Cutaneous Inflammation in the Mouse, After Inoculations of Different Human Pathotypes of Borrelia burgdorferi sensu stricto

The skin is an essential organ in the development of Lyme borreliosis because Borrelia is inoculated and multiplies before spreading in the body to reach the target organs: the joint, the nervous system and the skin at a distance.

Different human clinical isolates of B. burgdorferi ss

(pathotypes), with different virulence factors of RSTs type (16S-23S rRNA intergenic spacer type) were selected. Inflammatory responses at the cutaneous level in a murine model were compared according to the protocol described below to determine whether the immunity of the skin played a role in the organotropism of bacterial strains. The mouse is indeed a model of choice for understanding the pathogenicity mechanisms of B. burgdorferi if [28].

2.1 Material and methods

2.1 .1 Mice and bacterial strains

C3H / HeN mice three to four weeks old were used (Charles River Laboratories, L'ArbresIe, France). Strains of Borrelia burgdorferi sensu stricto have been isolated from patients with different clinical manifestations: the PBre strain (RST1) of an erythema migrans (EM) (single lesion - Germany), the MR726 strain (RST3) of a migrating erythema multiple (United States), cerebrospinal fluid strain 1808/03 (RST1) (Slovenia) and cerebrospinal fluid strain 297 (RST2) also (United States). The clone Borrelia c297 / 4 was selected by culture on solid BSK medium [19]. All strains were cultured in BSK-H complete medium (Sigma) at 33 ° C and used at low passage (<7). Borrelia were counted and viability was verified by dark field microscopy.

2.1 .2 Monitoring the infection of the mouse

The mice were infected with 10 ³ spirochaetes in 0.1 ml of BSK medium intradermally in the dorsolumbar region. The control mice were injected with an equal volume of sterile BSK medium and maintained under the same conditions as the infected animals. Evaluation of arthritis was performed weekly by measuring the thickness of both tibio-tarsal joints with a metric vernier caliper. Joint measures provided an indication of the severity of arthritis. Serology was performed as described in Kern et al. [29].

At different times after the start of the experiment (Oh, 5h, 24h, 3j, 5j, 7j, 15j and 30j after infection) the mice were killed by overdose of isoflurane gas. About 1 cm of skin was collected at the site of inoculation and stored in Trizol (registered trademark) (Invitrogen).

The ear, the base of the heart, the bladder and the tibiotarsal joints of Each mouse was aseptically collected and divided into two parts, for PCR and Borrelia culture. The organs of the uninfected mice were collected under the same conditions as the positive mice.

2.1 .3 Detection of B. burgdorferi in mouse organs

For the detection of spirochaetes by culture, the organs removed were placed in 6 ml of BSK-H medium containing 30 g of rifampicin (BioRad). The tubes were maintained at 33 ° C, and the presence of spirochaetes was examined weekly by darkfield microscopy.

For PCR, DNA was extracted from the organs of each mouse on a MagNA Pure system (Roche Diagnostics, France), using a MagNA Pure LC wide-volume isolation kit after external lysis. The heart, the bladder, the ear and the skin were placed in 500 μl of lysis buffer containing proteinase K. Other samples were treated with external lysis by collagenase A and then proteinase K. the DNA samples were finally eluted in 100 μl of elution buffer. Ten μί of Borrelia DNA was used as a positive control for detection. Qualitative amplification was performed as described in Woods et al. [30]., Targeting the flagellin gene.

2.1 .4 Quantification of spirochete burden and inflammatory genes in the skin of the mouse

At the inoculation site, quantification of the B. burgdorferi specific flagellin gene was performed on a LightCycler system (Roche Diagnostics, France). The primers used to amplify the fia gene were those described in Kern et al. [29].

To measure inflammation at the inoculation site, total RNA was extracted from 10 mg of mouse skin using Trizol reagent according to the manufacturer's indications (Invitrogen). The samples were treated with DNAse (Ambion, USA) and then a first strand of cDNA was synthesized from 1 g of total RNA using SuperScript II reverse transcriptase. (Invitrogen Life Technologies). Quantification of gapdh was performed as an internal standard. Relative expression levels were calculated using an infected animal as a calibrator. Amplification and detection were performed with an ABI 7000 system with the following thermal profile: 95 ° C for 10 minutes, 50 cycles of 95 ° C for 15 seconds, 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 B. buradorferi strain 297, wild-type strain and virulent clone c297 / 4

The cultures of B. burgdorferi 297, the wild strain and the virulent c297 / 4 clone 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 Gene dynamics of B. buradorferi 297. of the wild-type strain and virulent clone c297 / 4 in the skin of mice at the site of inoculation

At different times (0, 5, 24, 3, 5, 7, 15 and 30 days after infection), skin samples were taken from each mouse at the site of inoculation. Total RNA was purified using Trizol reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNAs were determined by measuring the optical density at A260 and A280. The samples were then treated with gDNAse (QIAGEN) to remove the contamination with DNA. Total extracted RNAs were submitted to the Quantiscript Reverse Transcription (QIAGEN) to produce the cDNA. CDNA was used to quantify the ospC and bbk32 genes. For B. burgdorferi C297 / 4, selected genes corresponding to cell envelope proteins were retained for RT-PCR. Relative expression levels were calculated using the AACt method with flagellin as the internal standard. The amplification and detection were performed with an ABI 7500 system with the following thermal profile: 95 ° C for 10 minutes, 50 cycles of 95 ° C for 15 s, at 50 ° C for 30 s and 60 ° C for 1 min. Each amplification condition was compared to day 3 for relative quantization. Correlation factors were calculated by comparing the cDNA amplification of each kinetic point of the native strain to the cDNA amplification of each point of the hypervirulent clone. Then, the curve obtained for the clone, was normalized by these factors to obtain a second curve, representative of the wild-type strain, and quantitatively comparable to the clone.

2.1 .7 Statistical Analysis

Each experiment was performed at least three times. For each RT-PCR, at least two extractions were performed for each mouse in each experiment, with two to three mice for each point. 2.2 Results

2.2.1 Transmission and diffusion of different pathotypes of B. buradorferi ss in mice

All strains studied showed a similar pattern of diffusion. Borrelia was detected at day 3 by PCR in the skin, at the site of inoculation, for all strains. They spread rapidly in the joint, where they were first detected at day 5 or 7, then to the heart and bladder at day 5, 7 or 15, and the slowest spread occurred in the ear (skin away from the site of inoculation) ,. The PBre pathotype, isolated from erythema migrans (EM), spread more slowly to the heart and bladder compared to the others. In ELISA, all mice were positive for Borrelia antigens 15 days after bacterial inoculation.

2.2.2 Quantification of Borrelia pathotypes and measurement of inflammation at the site of inoculation The bacterial load of the skin was measured. All strains multiplied intensively on day 7, but no significant difference was observed between the strains tested.

The inflammatory profile in the skin of the mice was compared for these different strains of B. burgdorferi ss. Antimicrobial peptides (AMPs), markers of the innate immunity of epithelia, were measured. The PBre (EM) strain induced a significant amount of cathelicidin with a peak on day 3. The MR726 (MEM) strain strongly induced defensin mBD-3. Wild type strain 297 (CSF) showed a peak of mBD-3 at 24 h while strain 1808/03 (CSF) induced a negligible amount of all three MPAs tested. The induction of additional pro-inflammatory molecules was then measured: TNF-α, IL-6, IL-22 and the chemokine MCP-1. For each of them, a peak of induction of TNF-α and / or MCP-1 was observed on day 7. The MR726 strain isolated from a lesion of the MEM, induced the stronger inflammatory profile at the level of mouse skin with a peak of MCP-1 (150 times) at day 7.

2.2.3 Specific analysis of the inflammation induced by B. burgdorferi ss 297 wild strain and its hypervirulent clone

Borrelia infection could be initiated by a heterogeneous population of Borrelia in the vertebrate host. A clone C297 / 4 from B. burgdorferi ss 297 was selected in the laboratory for its rapid diffusion and its neurological manifestations in mice [31]. The virulent clone C297 / 4 caused inflammation of the skin with a greater induction of defensins, MBD-14, and cathelicidin compared to the wild-type strain. A very high induction of MCP-1 and IL-6, approximately 100-fold more induction, was observed for clone C297 / 4 compared to the wild-type strain.

The results of the wild strain 297 and the hypervirulent clone were also compared in the C3H / HeN mouse. The hypervirulent clone spread more rapidly to the joint, while diffusion to other organs was similar to that of the wild-type strain. Quantification of the bacterial load in the tissues confirmed the intense multiplication occurring in the skin at day 7 regardless of the strain used, but no significant difference was observed between the hypervirulent clone and the wild strain 297.

2.2.4 Proteomic characterization of B. buradorferi ss, wild type 297 strains and clone hvpervirulent

The protocol presented in Examples 1 .2.1 and 1.2.2 above has been applied.

A total of 887 proteins were identified with 848 c297 / 4 proteins and 777 proteins in the wild-type strain. We observed an overlap of 738 proteins which represents 83% of the total number. 1 10 proteins are specific for the hypervirulent clone.

2.2.5 Comparative expression of the specific proteins of the wild-type and hvpervirulent clones of B. buradorferi 297 in the skin of the mouse by RT-PCR.

The kinetics of expression of OspC and BBK32, two important proteins in Borrelia transmission, were performed for wild-type and c297 / 4 strains. Both strains show a first peak of OspC expression at day 5, while a peak of BBK32 expression was observed at day 7. Then, Borrelia surface proteins were selected from the 1 10 proteins. specific to the hypervirulent clone, and their expression was followed during cutaneous inflammation in C3H / HeN mice. Three genes are highly expressed in both strains, bb0304, bb0213 and bb0347 with peak expression at day 5 for the hypervirulent clone and at day 7 for the wild-type strain.

Example 3 Selection of vaccine candidates and determination of the immunogenic effect

The different proteins are tested in a mouse model

C3H / HeN to see their expression on the skin, during the transmission of the bacteria. Indeed, the cutaneous interface seems to play a key role in the selection of certain bacterial populations (Brisson et al., 201 1 [32]). The skin of intradermally infected mice is removed at 3, 5, 7 and 15 days. After designing the specific primers for each of the proteins, the RT-PCR technique is used to monitor the expression of these proteins in the skin. 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., 201 1 [15]) and expressed in E. coll. They are inoculated intradermally with the mouse and the antibody level is measured by Elisa. Indeed, the antibody response appears essential for measuring a protective effect during Lyme borreliosis (Embers and Narasimhan, 2013 [34]). Their protective effect is tested by challenge with Borrelia inoculated with the syringe, or better with ticks infected with Borrelia. Vaccinated and challenged mice are then dissected and their organs cultured or tested in PCR to measure the absence of Borrelia (Kern et al., 201 1

[29]).

Five proteins were selected for in vivo tests in mice, namely the three "hypothetical protein" proteins only detected in the virulent clones and common to the three species of Borrelia (SEQ ID NO: 10 (BB0566), 13 (BB0173) and 16 (BB0722) of Borrelia burgdorferi ss and corresponding sequences SEQ ID NO: 11 (BAPKO0596), 14 (BAPKO0175) and 17 (BAPKO0766) respectively of B. afzelii) and 12 (BG0576), (BG0172) and 18 (BG0172). BG0744) of B. garinii)), the RpoN protein (SEQ ID NO: 41 (BB0450) from Borrelia burgdorferi ss and the corresponding sequence SEQ ID NO: 42 (BAPKO0472) B. afzelii)) and the Gnd protein (SEQ ID NO Borrelia burgdorferi ss 77 (BB0561) and the corresponding sequence SEQ ID NO: 42 (BAPKO0590) B. afzelii)). At the same time, in the skins of infected mice 7 days after intradermal inoculation, all Borrelia proteins expressed in the skins of infected mice were analyzed by a non-targeted proteomic approach. Indeed, 7 days corresponds to a peak of intense multiplication of bacteria after intradermal inoculation and therefore probably plays a key role in the initiation of bacterial infection (Kern et al., 201 1 [29]). The strategy consisted of extracting the proteins contained in the infected skin, prefracting them with electrophoresis gel and then identifying them by liquid chromatography coupled with tandem mass spectrometry (Ge-LC-MS / MS strategy).

3.1 Materials and method

3.1 .1 Inoculation of bacteria and removal of infected skin

Three to four week old C3H / HeN mice were purchased from Charles River Laboratories (L'ArbresIe, France).

The inventors have been particularly interested in the strain B. burgdorferi ss 297, isolated from cerebrospinal fluid in the United States.

(Sterre et al., 1893 [35])

Different clones of B. burgdorferi ss were obtained by culturing on solid BSK medium ([19]). The clone 297c4 was selected for its speed of dissemination in the mouse and its cerebral localization in particular.

All Borrelia strains were cultured in BSK-H (Sigma) medium at 33 ° C and used at low passage (<7) for mouse infection. Spirochetes were counted and viability was verified using the dark-field microscope. The mice were infected with 10 ³ spirochaetes in 0.1 ml BSK by intradermal injection into the thoracic dorsal region.

At various points after inoculation (3d, 5d, 7d, 15d), the mice were killed by isoflurane. A 1 cm area of mouse skin was collected at the site of inoculation and stored in Trizol (Invitrogen) for RT-PCR analyzes. For the quantification of Borrelia in the skin, the sample is kept dry at -80 ° C.

3.1.2 PCR quantification of skin infected with Borrelia

At the site of inoculation, detection of the presence of B. burgdorferi ss was performed by PCR targeting the flagellin gene on a LightCycler system (Roche Diagnostics, France). The primers used to amplify the fia gene are those previously described ([29]).

3.1 .3 RT-PCR on mouse skins for gene detection

At different points in the kinetics, skin samples were taken from each mouse at the site of inoculation. Total RNA was purified using Trizol reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNA was determined by measuring A260 and A280. The samples were then treated with gDNAse wipeout (QIAGEN). Total RNA extracted was synthesized into cDNA using Quantiscript Reverse Transcription (QIAGEN). CDNA was used to quantify the bbk32 genes (positive control). For B. burgdorferi ss 297 and 297c4, the genes corresponding to the three common proteins and RpoN and Gnd were tested in RT-PCR using the primers described in Table 3 below.

Table 3: Primers used for RT-PCR

Proteins SEQ ID NO Sequences

93 GB - AGG CTC GAA GGA GAG CTT GT

BB0566

94 R - AAA CAT TK CGG ATG GAT ACT CAA

95 E - GCT GAT TT GCC AGC GAG CTT A

BB0722

96 R - TCG GTC CAA ATA CTT CCG TAA CC

97 E - TCG CCT AGT ATG GGG GCT GTT

BB0173

98 R - AGC AGA ACC ATT GCC AAG ATC C

99 F - AAG TGA AAA CCC CCA AAA ACA AAA A

RpoN

100 R - TTG CTC CAC CAA CAG AGC TAA AAA G

101 GB - GGA ATG AAG GCG ATC TTT CAG GG

Gnd

102 R - GTC GGC AAA GGA ATC CCA ATT TCCA Relative expression levels were calculated using the flagellin method AACt as internal standard. Amplification and detection were performed with an ABI 7500 system with the following thermal profile: 95 ° C for 10 min, 50 cycles of 95 ° C for 15 sec, and 60 ° C for 1 min. Each amplification condition was compared to day 3 for relative quantization.

3.1.4 Proteomic analysis of infected skin

Biopsies were selected based on PCR quantification. Fragments of approximately 4 mg were cut out and the proteins were extracted into 200 μl Laemmli buffer and then assayed. Proteins (50 g) were pre-fractionated on SDS-PAGE electrophoresis gel and the migration tracks were excised and processed as described in Example 1. The tryptic peptides were analyzed by nanoLC-MS / MS using the nanoLC-Chip / MS system coupled to the amaZon ion trap, as described in Example 1. The spectra of MS and MS / MS were acquired with the same parameters and the searches were carried out in the same way except for the databanks. In this case, the searches were carried out in databases, consisting of sequences of B. burgdorferi ss B31 and mice, downloaded from the database NCBInr and UniProtKB-SwissProt, respectively (B. burgdorferi B31: August 16 2012; mouse: April 19, 2013).

3.2 Results

3.2.1 Borrelia multiplication peak at day 7;

The quantification of Borrelia in the skin at different points after intradermal inoculation reveals an intense multiplication of bacteria at day 7 and this regardless of the strain of Borrelia tested

(Figure 1 ). 3.2.2 Kinetics of expression of certain genes in the skin

The RT-PCR expression profile of the protein BB0566 (SEQ ID NO: 10), a protein common to all three Borrelia species: B. burgdorferi ss, B. afzelii and B. garinii, in the skin of mice during the early transmission of the bacteria is shown in Figure 2.

These results show that the protein of sequence SEQ ID NO: 10 is strongly overexpressed in the skin of mice from the fifth day after inoculation for the two strains 297 and 297c4.

3.2.3 Proteomic analysis of the skin of infected mice at day seven after intradermal inoculation

In cutaneous biopsies of infected mice, an average of 1350 mouse proteins were identified. Among the Borrelia proteins detected, the RpoN and Gnd proteins were identified which confirms the expression of these proteins in the skin seven days after inoculation and their potential role during the early transmission of the bacterium.

Example 4: Vaccine test

The dose of recombinant proteins to be administered is determined according to a prior dose-effect study well known to those skilled in the art, generally between 1 and 500 g. Depending on the vaccination protocol in the dog, ideally, two administrations will be performed between 2 to 4 weeks apart and an annual reminder. The adjuvant is chosen according to its ability to stimulate the humoral response and / or the cellular response. The person skilled in the art knows which adjuvant to choose to effectively stimulate the humoral response and / or the cellular response. The administration of the vaccine is carried out intradermally, subcutaneously or intramuscularly, preferably intramuscularly or subcutaneously. List of references

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[3] Radolf JD et al. Of ticks, mice and men: Understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol. 2012 Jan 9; 10 (2): 87-99.

[4] Stanek G et al. Lyme borreliosis. Lancet. 2012 Feb 4; 379 (9814): 461-73. doi: 10.1016 / S0140-6736 (1 1) 60103-7.

[5] Little SE et al. Lyme borreliosis in dogs and humans in the USA. Trends Parasitol. 2010; 26: 213-8.

[6] Hanson MS, Edelman R. Progress and controversy surrounding vaccines against Lyme disease. Expert Rev Vaccines. 2003; 2: 683-703.

[7] LaFleur RL et al. Bacterin that induces anti-OspA and anti-OspC borreliacidal antibodies provides a high level of protection against canine Lyme disease. Clin Vaccine Immunol. 2009; 16: 253-9.

[8] Töpfer KH, Straubinger RK. Characterization of the humoral immune response in dogs after vaccination against the Lyme borreliosis agent A study with five commercial vaccines using two different vaccination schedules. Vaccinated. 2007; 25: 314-26.

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[12] Peppas NA and Carr DA, Impact of Absorption and Transport on Intelligent Therapeutics and Nano-scale Delivery of Protein Therapeutic Agents Chemical Engineering Science, 64, 4553-4565 (2009).

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[19] From Martino et al., Enhanced culture of Borrelia garinii and Borrelia afzelii strains has a solid BSK-based medium in anaerobic conditions. Res. Microbiol. 2006, 157, 726-729.

[20] Laemmli, UK (1970) "Cleavage of structural proteins during the assembly of bacteriophage T4" Nature 227, 680 - 685. [21] Candiano et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25, 1327-1333.

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Claims

1. Vaccine composition comprising:

at least one Borrelia burgdorferi polypeptide ss selected from SEQ ID NO: SEQ ID NO: 10, 13, 2, 8, 9, 19, 25, 29, 33, 35, 43, 45 and 85.

2. Vaccine composition according to claim 1, wherein the at least one Borrelia burgdorferi ss polypeptide is selected from SEQ ID NO: 10.

3. Vaccine composition according to claim 1 or 2, comprising in addition to the at least one polypeptide of claim 1, at least one polypeptide of Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii selected from SEQ ID NO: 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.

4. A composition according to any one of claims 1 to 3, comprising in addition to the at least one polypeptide of claim 1, at least one other Borrelia burgdorferi ss, Borrelia afzelii or Borrelia garinii polypeptide selected from groups (c1 ), (c2) and (c3),

said group (d) comprising SEQ ID NO: 19 to 28,

said group (c2) comprising SEQ ID NO: 29 to 46 and 73 to 86,

said group (c3) comprising SEQ ID NO: 47 to 72 and 87 to 92,

provided that if said at least one polypeptide of claim 1 is:

is SEQ ID NO: 2, 8 or 9, said at least one other polypeptide is included in group (c3), or is at least one other polypeptide is included in any one of the groups (c1), (c2) and (c3).

The composition of any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier.

6. Vaccine composition according to any one of claims 1 to 5, comprising an adjuvant.

7. Vaccine composition according to any one of claims 1 to 6, comprising at least one other active ingredient.

8. Vaccine composition according to any one of claims 1 to 7 for use as a medicament.

9. Vaccine composition according to any one of claims 1 to 7, for use in the prevention of Lyme disease.