EP3801608A1

EP3801608A1 - Multi-epitopic peptide compounds and vaccines against leishmaniasis

Info

Publication number: EP3801608A1
Application number: EP19730700.2A
Authority: EP
Inventors: Rachel BRAS GONÇALVES; Jean-Loup Lemesre; Amel Garnaoui; Elodie PETITDIDIER-LESIN
Original assignee: Institut de Recherche pour le Developpement IRD; Institut Pasteur de Tunis
Current assignee: Institut de Recherche pour le Developpement IRD; Institut Pasteur de Tunis
Priority date: 2018-05-30
Filing date: 2019-05-29
Publication date: 2021-04-14
Also published as: FR3081870B1; FR3081870A1; BR112020024211A2; US11559575B2; WO2019243018A1; US20210213114A1; TN2020000219A1

Abstract

The invention relates to multi-epitopic peptide compounds obtained from PSA, HwB and LmLRAB proteins of Leishmania as well as to pharmaceutical compositions and vaccines for use against one or more leishmaniases.

Description

MULTIEPITOPIC PEPTIDE COMPOUNDS AND VACCINES AGAINST

LEISHMANIOSIS

FIELD OF THE INVENTION

The present invention relates to peptide compounds comprising epitopes, pharmaceutical compositions comprising such peptide compounds, nucleotide sequences coding for the epitopes included in these peptide compounds or for said peptide compounds, expression vectors comprising at least one of these sequences nucleotides, diagnostic reagents comprising said peptide compounds, as well as prophylactic and / or therapeutic vaccines, intended for use against one or more of the leishmaniases.

PRIOR ART

The cutaneous (LC), cutaneous-mucous (LCM), diffuse and visceral cutaneous (LV) forms of leishmaniasis are among the most serious parasitic infections affecting humans. They constitute a real public health problem, in particular in Latin America, Asia, Africa and southern Europe.

In addition to patients with symptoms of leishmaniasis, there is a high number of asymptomatic individuals who are infected but untreated because they have not been diagnosed. The southern Mediterranean region is an endemic area for L. infantum. In this region, more than 20% of the blood donor population is infected, while only a few cases of clinical VL are diagnosed. The ratio of asymptomatic individuals / individuals symptoms are even higher in areas endemic to L. donovani. In fact, in these zones, this ratio is between approximately 9/1 and approximately 3/1.

The elimination of leishmaniasis must take into account asymptomatic individuals, who are considered as potential reservoirs of leishmaniases.

Although current chemotherapeutic treatments such as, for example, treatments using AmBisome ™, have made considerable progress, a therapeutic approach cannot be applied to asymptomatic carriers.

Vaccination is the most suitable way to interrupt the transmission of Leishmania parasites and eliminate leishmaniasis.

To date, canine vaccines are used. Examples include the CaniLeish ™, Leish-Tec ™ or Leishmune ™ vaccines. However, no vaccine for human application is available.

Such a human vaccine would, however, have significant health and socio-economic consequences. In particular, coupled with the use of a canine vaccine, its use would lead not only to the reduction of the canine reservoir of visceral leishmaniasis, but also to a significant reduction in the most disabling and even fatal human pathologies. In fact, as with malaria, leishmaniasis has a significant impact on populations and the economy, and its prophylaxis should significantly improve the socio-economic context of the most seriously affected countries.

We imagined developing a human vaccine by transposing a canine vaccine such as CaniLeish ™ to humans, this canine vaccine being the subject of patent document W09426899, or using only one of the major immunogens of the vaccine, subject of patent document W02015079420, or using only peptide derivatives of this major immunogen, subject of the patent document

W02014102471, which in particular discloses the following peptides:

SEQ ID N ° B9: T-N-T-L-A-V-L-Q-A-F-G-R-A-I-P-E-L-G-

K-K-W

SEQ ID N ° B10 E-G-Y-F-V-T-D-E-K-T-G-L-V-Y-R-D-G-G- V-A-A-A-S-S-G

SEQ ID N ° B11 T-P-E-Q-R-T-N-T-L-T-V-E-L-G-K-K-W-I-

G

SEQ ID N ° B12: T-L-P-E-M-P-V-G-V-P-E-M-P-A-G-V-D-Y SEQ ID N ° B13: A-R-G-R-E-G-Y-F-L-A-R-G-A-R-G-R-E-G-Y-E-G-Y-E-G

However, the transposition of the active principle of this vaccine or of the major immunogen alone to human medicine is not possible given the industrial constraints relating, in particular, to the production of human vaccines, and the costs involved. The use alone of these peptide derivatives of the major immunogen is also not possible since they have an insufficient vaccination coverage rate for the populations concerned and an in vitro efficacy which is too weak to stimulate human cells and to produce cytokines. Thl type like IFN-g, even in 2mM combinations for each peptide. In addition, other arguments are also in favor of the development of a specific vaccine for the prevention of human leishmaniasis. Indeed, it has been shown that the cure of this disease is generally associated with the development of immunity to reinfection. In recovered individuals, a cellular immune response specific to parasitic antigens is observed both in vivo, during the delayed hypersensitivity reaction in response to the leishmania skin test, and in vitro, during stimulation of the mononuclear cells of the peripheral blood (PBMC) of these individuals, with Leishmanian antigens.

SUMMARY OF THE INVENTION

In view of the above, a technical problem which the invention proposes to solve is to develop peptide compounds comprising new epitopes, pharmaceutical compositions and diagnostic reagents comprising such peptide compounds, having a strong immunogenic power against leishmaniasis, and allowing the development of prophylactic and / or therapeutic vaccines intended to be used effectively against one or more of the species of effective leishmanias, which are, financially, accessible to the populations concerned.

The solution of the invention to this problem posed has for first object a peptide compound comprising at least 2 epitopes contained in the protein sequences chosen from the PSA, H2B or LmLRAB proteins of Leishmania having a sequence chosen from the sequences SEQ ID No 1 at 21, the sequences SEQ ID N ° 24 to 64, the sequences SEQ ID No 67, 68, 70 to 72, 74 to 76, the sequences SEQ ID No 78 to 95, sequences SEQ ID No 98 to 136, sequences SEQ ID No 139 to 156, and sequences SEQ ID No 158 to 184, as well as its analogous, mutein and homologous derivatives, said epitopes possibly being separated by a peptide spacer comprising at least 1 amino acid.

The second object is a specific antibody and immuniserum containing it directed against epitopes of the abovementioned peptide compounds.

Its third object is a pharmaceutical composition comprising at least one peptide compound as above.

Its fourth object is a composition comprising at least one peptide compound as above, for the manufacture of a medicament or of a vaccine, of an in vivo or in vitro diagnostic reagent for induction or diagnosis in mammals, activation of cell-mediated immunity dependent on Th1-type lymphocytes and / or effector humoral immunity.

Its fifth object is a prophylactic and / or therapeutic vaccine intended to be used against one or more of the leishmanias chosen from Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania major , Leishmania (Viannia) hraziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, and / or Leishmania (Viannia) peruviana, comprising at least one peptide compound as above.

Its sixth object is a nucleotide sequence coding for epitopes included in the above peptide compounds or for said peptide compounds.

Its seventh object is an expression vector comprising at least one nucleotide sequence as above, as well as the means necessary for their expression.

Its eighth object is a diagnostic reagent comprising a peptide compound as above.

Advantageously, the peptide compound is a peptide compound in which said analogous derivatives, muteins and homologs of the epitopes, having immunogenic power, have a percentage of sequence identity of at least 50%, preferably at least 75% with the sequence of said epitopes; - the peptide spacer comprises 1 to 8 amino acids; the peptide compound comprises 2, 3 or 4 of said epitopes; a first epitope is chosen from said epitopes contained in the sequence of a first protein of the proteins chosen from PSA, H2B or LmLRAB, a second epitope is contained in the sequence of a second protein, different from the first protein, and chosen from PSA, H2B or LmLRAB; the peptide compound is a compound in which the epitopes are chosen from epitopes of sequence SEQ ID No 22, 23, 65, 66, 69, 73,

77, 96, 97, 137, 138, 157 and 185; the peptide compound comprises at least 3 epitopes contained in the sequences of different proteins chosen from PSA, H2B and LmLRAB; - The composition comprises at least one peptide compound as above and is intended to be used in prophylactic and therapeutic vaccination directed against leishmanias; - the leishmanias are the Leishmania donovani, the Leishmania infantum, the Leishmania chagasi, the Leishmania mexicana, the Leishmania amazonensis, the Leishmania venezuelensis, the Leishmania tropica, the Leishmania major, the Leishmania aethiopica, the Leishmania (Viannia) Viannia) guyanensis, Leishmania (Viannia) panamensis and / or Leishmania (Viannia) peruviana; the composition comprises at least one peptide compound as above, for the manufacture of a medicament or of a vaccine, of a reagent for in vivo or in vitro diagnosis for the induction or the diagnosis in mammals of activation of cell-mediated immunity dependent on Thl-type lymphocytes and / or effector humoral immunity; - The vaccine comprises on the one hand, at least one peptide compound chosen from SEQ ID No. 22, 23, 69, 73 and 77, as well as its analogous derivatives, muteins and homologs; and on the other hand, at least one peptide compound chosen from the sequences SEQ ID No. 65, 66, 96, 97, 137, 138, 157 and 185, as well as its analogous derivatives, muteins and homologs; - The vaccine comprises on the one hand, at least one or two peptide compounds chosen from SEQ ID No. 22, 23, 69, 73 and 77 as well as its analogous derivatives, muteins and homologs; and on the other hand, at least one, two or three peptide compounds chosen from the sequences SEQ ID No. 65, 66, 96, 97, 137, 138, 157 and 185 as well as its analogous derivatives, muteins and homologs; the vaccine comprises, in combination, the following multiepitopic peptide compounds: SEQ ID No 23, 66, 73, 77, 97, 138, 157 and 185 as well as its analogous, mutein and homologous derivatives; SEQ ID No 23, 66, 157 and 185 as well as its analogous derivatives, muteins and homologs; SEQ ID No 66, 97, 138, 157 and 185 as well as its analogous derivatives, muteins and homologs; or SEQ ID No 66, 157 and 185 as well as its analogous derivatives, muteins and homologs; and the vaccine also comprises an adjuvant chosen from adjuvants of classes TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, saponins and their derivatives QA21, quilA or QS21, oil in water or water in oil emulsions, polysaccharides , cationic liposomes, virosomes or polyelectrolytes and immunomodulators chosen from sandfly saliva proteins, cytokines, peptides and heat shock proteins; the vaccine is intended for subcutaneous, intradermal, intramuscular, parenteral, endonasal, mucosal or oral administration.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood on reading the following non-limiting description, drawn up with reference to the appended drawings, in which:

FIG. IA represents the localization of the HLA-I and HLA-II epitopes of sequences SEQ ID No. 1 to 66 on the sequence of the protein H2B of L. major (GenBank accession No. AAK21263), and FIGS. IB and IC represent the localization of the main HLA-I epitopes of the sequences SEQ ID No 22 and 23 (Figure IB), and the localization of SEQ ID No 65 and 66 (Figure IC) on said sequence of the protein H2B of L. major;

FIGS. 2A, 2B and 2C represent the localization of the HLA-I epitopes of sequences SEQ ID N ° 67-68, 70-72 and 74-76 (FIG. 2A), the localization of the HLA-I and HLA-II epitopes of sequences SEQ ID N ° 78 to 97 (Figure 2B), and the location of the HLA-I and HLA-I I of sequences SEQ ID No. 98 to 138 (FIG. 2C) on the sequence of the LmLRAB protein of L. major (GenBank accession No. XP_001685071) and FIG. 2D represents the location of the sequences SEQ ID No. 96, 97, 137 and 138 (Figure 2D) on the sequence of said LmLRAB protein from L. major;

FIGS. 3A and 3B represent the location of the HLA-I and HLA-II epitopes of sequences SEQ ID No. 139 to 157 (FIG. 3A) and the Location of the HLA-I and HLA-II epitopes of sequences SEQ ID No. 158 to 185 on the sequence of the PSA protein of L. infantum (GenBank accession N °

ACY70941) and FIG. 3C represents the location of the sequences SEQ ID Nos. 157 and 185 on this sequence of the PSA protein of L. infantum;

FIG. 4 is a table which shows the estimated affinities for the HLA class I and HLA class II alleles of the epitopes within the peptides of the sequences SEQ ID No 22, 23, 65, 66 (FIG. 4A), 69, 73, 77, 96, 97,

137, 138 (Figure 4B), and 157, 185 (Figure 4C);

FIG. 5 is a table which shows the coverage of the world population estimated with a combination of the peptide compounds according to the invention (FIG. 5A), or with the combination of the peptides SEQ ID No. B9, B10, B11,

B12 and B13 (FIG. 5B), which shows a very high added value of the peptides according to the invention;

FIG. 6 is a table which shows that the levels of IFN-g secreted by PBMC (Peripheral Blood

Mononuclear Cells - Mononuclear Cells of Peripheral Blood) of individuals cured of cutaneous leishmaniasis and stimulated in vitro by peptide compounds of the present invention according to a short 10-day protocol with stimulation on D0 and addition of recombinant IL-2 to Jl, J4 and J7. IFN-g levels can reach more than 2700 pg / mL with a combination peptide compounds according to the invention, which shows a very high added value of these peptides according to the invention. The compounds used in these mixtures were obtained by chemical synthesis with the addition of a palmitoylated tail at the amino-terminal end and without modification of the carboxy-terminal end;

FIG. 7 is a table which shows that the levels of IFN-g secreted by the PBMCs of individuals cured of cutaneous leishmaniasis and stimulated in vitro by mixtures of peptide compounds of the present invention according to a short 10-day protocol with stimulation at OJ and addition of recombinant IL-2 at J1, J4 and J7. The compounds used in this mixture were obtained by chemical synthesis with the addition of a palmitoylated tail at the amino-terminal end and amidation of the carboxy-terminal end. The levels of IFN-g obtained with a combination of the peptide compounds according to the invention also shows a high added value of the compounds according to the invention;

FIG. 8 shows the evaluation of the immunogenicity properties of peptide compounds of the invention in naive individuals, persons who have never been in contact with the Leishmania parasite. This evaluation is an important factor in predicting the efficacy of a peptide vaccine (Kwok WW, et al 2012, Frequency of epitope-specific naive CD4 (+) T cells correlates with immunodominance in the human memory directory; Castelli FA, et al 2007, Differential capacity of T oeil priming in naive donors of promiscuous CD4 + T oeil epitopes of HCV NS3 and Core proteins). Immunogenicity is evaluated in vitro by measuring the frequency of preexisting naive T cells and the amplitude of the naive T repertoire specific for each compound. The evaluation is carried out on the basis of blood samples from naive individuals and according to a long protocol (Castelli FA, et al. 2007 cited above) bringing purified T lymphocytes into contact with autologous dendritic cells previously incubated with the peptides of the invention. The combination of peptides used is composed of: SEQ ID No 23, SEQ ID No 66, SEQ ID No 73, SEQ ID No 77, SEQ ID No 97, SEQ ID No 138, SEQ ID No 157, SEQ ID No 185 , SEQ ID No Bll, SEQ ID No B12 and SEQ ID No B13. The compounds were synthesized chemically without modification in amino-terminal and with amidation (NH2) in carboxy-terminal (Peptide-NH2). After 3 successive stimulations at 7-day intervals of the CD4 + T cells of a naive individual (MPL10; French blood establishment in Toulouse), an ELISPOT IFN-g was carried out with the cells in the presence or absence of the mixture of peptides, or in the presence of each compound individually. Six specific lines were highlighted (p-value between 0.0269 and 0.0489 depending on the specific line). Each positive line responds to at least one of the peptides of the present invention (Figure 8). The MPL10-3 line responds to the peptides SEQ ID No73 and SEQ ID Nol57. The MPL10-4 line responds to peptides SEQ ID No 23, SEQ ID No 73, SEQ ID No77, SEQ ID No 97, SEQ ID No 138 and SEQ ID Nol57. The MPL10-7 line responds to peptides SEQ ID No 77, SEQ ID No97 and SEQ ID Nol57. The MPL10-8 line responds to peptides SEQ ID No 66, SEQ ID No 73, SEQ ID No97, SEQ ID Nol38 and SEQ ID Nol57. The MPL10-10 line responds to SEQ ID No66, SEQ ID No 73, SEQ ID No77, SEQ ID No 97, SEQ ID No 138, SEQ ID Nol57 and SEQ ID No 185. And the MPL10-12 line responds to peptides SEQ

ID No 66, SEQ ID No 157 and SEQ ID No 185; and

FIG. 9 shows the evaluation of the immunogenicity properties of peptide compounds of the invention in the same naive individual (MPL10) but with compounds synthesized chemically with addition of a tail palmitoylated at the amino-terminal end and with an NH2 group at the carboxy-terminal end (PAL-peptide-NH2). The same combination of peptides as before was used. After 3 stimulations with the mixture of PAL-peptide-NH2 peptides, an IFN-g ELISPOT was carried out with the cells in the presence or absence of the mixture of peptides, or in the presence of each peptide individually. Four specific lines were highlighted (p-value = 0.0275 for all specific lines). The MPL10-PAL-2 line responds to the peptides SEQ ID No 22, SEQ ID No 66, SEQ ID No 73, SEQ ID No 157 and SEQ ID No 185 (Figure 9). The line MPL10-PAL-3 responds to the peptides SEQ ID No 23, SEQ ID No 66, SEQ ID No 73, SEQ ID No 77, SEQ ID No 97, SEQ ID No 138, SEQ ID No 157 and SEQ ID No 185.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the term "epitope" means a peptide compound or peptide defined by its sequence having approximately 8 to 15 amino acids.

According to the invention, the term "analogous derivatives" or "mutein derivatives" of a peptide compound means the biologically active derivatives of the reference molecules which exhibit the desired activity, namely the ability to stimulate a cell-mediated immune response . Generally, the terms "analogous derivatives" refer to compounds having a sequence and a polypeptide structure having one or more additions, substitutions and / or deletions of amino acids, compared to the peptide compounds defined above, in the since these changes do not destroy the immunogenic activity. According to the invention, the particularly preferred "analogs" include substitutions preservatives, that is to say substitutions or replacements without consequences on the function and the final structure of the protein. The term "mutein derivative" is intended to mean peptides having one or more elements imitating the peptide. Methods for preparing conventional analogs and muteins are known to those of skill in the art.

According to the invention, the term "homologous derivatives" means peptide compounds having a certain percentage of peptide identity. The term "identity" means that the amino acids of two compared peptide sequences exactly match. The percentage of identity is determined by a direct comparison of the sequences between two peptide compounds by aligning said sequences and by counting the exact number of mismatches between the two aligned sequences. Then divide by the length of the shortest sequence and multiply the result by one hundred. The percentage of identity can also be determined using computer programs well known to those skilled in the art. Thus, according to the invention, two peptide sequences are said to be "substantially homologous" with respect to each other, since they have at least 50%, preferably at least 70%, more preferably at least 75 %, more preferably at least 85%, more preferably at least 90% and more preferably at least 95% or more of sequence identity over a defined length of the peptide molecules.

The vaccine strategy according to the invention is intended to meet the need for a vaccine ensuring good coverage of the world human population and protecting against the main species of leishmaniasis. It is based on fragments antigenic peptides capable of activating, in a lasting way, the specific cellular immunity directed against these parasites. Peptide vaccination is based on the molecular and cellular bases of recognition of the antigen by T cells. The establishment of specific immunity depends, to a large extent, on the degradation and association of antigenic fragments, peptides, with molecules of the Major Histocompatibility Complex (MHC; HLA for Man). This association is made specific for a particular HLA molecule by amino acid residues constituting the anchoring patterns of the peptide. The complexes thus formed are recognized by T lymphocytes via a membrane receptor (TcR) and require a specific interaction with certain amino acids of the T epitope. T epitopes are ligands of HLA molecules with strong affinities or moderate. They are presented to CD8 + (cytotoxic) or CD4 + (helper) T cells by HLA molecules of class I or class II respectively. The formation of these tri-molecular complexes (TcR / HLA / peptide) is the prerequisite for the activation and expansion of specific T cells and therefore for the induction of a protective immune response during infection.

The vaccine strategy according to the invention is based on the identification and selection of immunodominant peptides carried by the sequences of the following specific proteins of Leishmania:

i) a virulence protein characterized as the major immunogen of the antigens excreted-secreted by leishmanias, namely the soluble PSA protein, which is common and very conserved within species of leishmanias, responsible for the various human infections; ii) a protein H2B, which is highly conserved among the Leishmania species, which is capable of inducing protection in the mouse model and a cellular response in humans, and of which the N-terminal region, which is the most divergent compared to mammalian histone proteins, is able to confer significant protection against a challenge with virulent parasites in BALB / c mice. The H2B protein is recognized by T lymphocytes from cured individuals of L. tropica or L. major, with an induction of high levels of IFN-g; and

iii) an LmLRAB protein, belonging to the RAB GTPases superfamily and highly conserved with other Leishmania species, capable of inducing significantly high levels of IFN-g in individuals cured of cutaneous leishmaniasis.

The interest of such peptides is multiple:

(i) they are very harmless since they avoid any infectious risk linked to the use of pathogenic agent or infectious agent;

(ii) they are chemically well defined and thus meet the pharmaceutical requirements for purity;

(iii) they facilitate the monitoring of the induced immune response (search for cytotoxic T lymphocytes (CTL) directed against a defined T epitope); and

(iv) their sequence can be modified in order to make them more immunogenic

Such peptides respond remarkably to all of the conditions mentioned in the preamble to the present application: reproducible, thermostable vaccine facilitating its conservation and transport, versatile, easy to produce at low cost in the areas endemic, making possible its large-scale use.

Advantageously, the epitopes or peptide compounds which are the subject of the invention are linked to carriers making it possible to make them more immunogenic. By way of nonlimiting examples of carriers, mention may be made of carrier proteins KLM (Keyhole limpet hemocyanin), lipopeptides of palmitoyl type, or their derivatives. The most common modifications of proteins by lipids are: isoprenylation, N-myristoylation, palmitoylation (or S-acylation) and glypiation. Isoprenylation and N-myristoylation are co-translational or immediately post-translational modifications and the group which is attached remains until the degradation of the protein. Palmitoylation is post-translational. This change is reversible and faster than the "turn over" of protein degradation: it can therefore be regulated. Glypiation is co- and post-translational. Palmitoylated peptides are particularly advantageous according to the invention, because these derivatives interact with the lipid components of the membrane of the target cells (macrophages, dendritic cells, neutrophils, etc.), promotes their penetration and transports them inside them. to then present them to the immune system.

The epitopes according to the invention also advantageously have one or more protective groups. Indeed, in order to improve the resistance to degradation, it may be appropriate to use a protected form of the peptide according to the invention. The form of protection is a biologically compatible form and is compatible with use in the field pharmaceutical. Numerous biologically compatible forms of protection can be envisaged, such as, for example, the acylation or acetylation of the amino-terminal end, or the amidation or esterification of the carboxy-terminal end, and so on. this is the case, for example, for the compounds used in FIG. 7 obtained by chemical synthesis with the addition of a palmitoylated tail at the amino-terminus and amidation of the carboxy-terminus. Thus, the invention also relates to an epitope as defined above, characterized in that it is in protected form. A protection based on a substitution on the amino-terminal can be used with an acetyl group, a benzoyl group, a tosyl group or a benzyloxycarbonyl group. Preferably, a protection based on amidation of the hydroxyl function of the carboxy-terminal end is used by a NYY group with Y representing an alkyl chain from C1 to C4, or esterification by an alkyl group. It is possible to protect both ends of the peptide compound.

The peptide derivatives according to the invention also relate to the amino acids and the peptides linked together by a pseudo-peptide bond. The term pseudo-peptide bond is understood to mean all types of bonds capable of replacing the conventional peptide bonds. In the field of amino acids, the geometry of the molecules is such that they can theoretically be in the form of different optical isomers. There is, in fact, a molecular conformation of the amino acid (aa) such that it deviates to the right the plane of polarization of light (dextrorotatory conformation or D-aa), and a molecular conformation of the amino acid (aa) such that it deviates to the left the plane of polarization of light (levorotatory conformation or L-aa). The natural amino acids are always of levorotatory conformation, consequently, a peptide of natural origin will consist only of amino acids of the L-aa type. However, chemical synthesis in the laboratory makes it possible to prepare amino acids having the two possible conformations.

From this basic material, it is thus possible to incorporate during the peptide synthesis both amino acids in the form of optical dextrorotatory or levorotatory isomers. Thus, the amino acids constituting the peptide according to the invention can be in the L-, D- or DL- configuration.

The epitopes and peptide compounds according to the invention can be obtained either by conventional chemical synthesis in solid phase or in homogeneous liquid phase, or by enzymatic synthesis, from constituent amino acids or their derivatives. The epitopes and peptide compounds according to the invention can also be obtained by fermentation of a strain of bacteria modified or not, by genetic engineering, or by extraction of proteins of animal or plant origin, preferably of plant origin, followed by 'a controlled hydrolysis which releases peptide fragments corresponding totally or partially to the epitopes and peptide compounds according to the invention.

The Applicant has been able to demonstrate that the different epitopes according to the invention are consensus sequences common to the main leishmania species and have a strong and medium affinity for all of the molecules of the MHC (Major Complex Histocompatibility) of mammals, and more particularly for all of the HLA molecules (HLA for human leukocyte antigens), mainly represented in the human populations most seriously affected by these conditions.

Indeed, in the context of a vaccination strategy for human populations exposed to Leishmanian infections and in order to meet the need for a vaccine ensuring good coverage of the world population and protecting against leishmaniasis, it is important to use fragments antigenics / peptides capable of activating, in a lasting way, the specific cellular immunity directed against the parasite.

To ensure good coverage of the world population and in view of the great variability of the HLA phenotype (major human histocompatibility complex) between individuals, the immunogenic antigenic fragments (peptides) of sufficient length must contain a series of epitopes capable of be presented by several types of HLA class I and II molecules.

HLA molecules are highly polymorphic. Indeed, there are more than 2500 HLA class I proteins (HLA-I) and more than 1000 HLA class II proteins (HLA-II). However, some of these HLA molecules, close in sequence and in spatial conformation, can present epitopes common to T cells. The grouping of several thousand HLA molecules is today described in a little more than twenty categories, called “HLA supertypes” presenting very conserved epitopes for each supertype. In addition to the development of peptide vaccines, there is the multiepitopic or polyepitopic approach (peptide containing several epitopes). This multi-epitopic approach is advantageous for developing a vaccine intended for the entire world population. Indeed, to ensure good coverage of the world population and in view of the great variability of the HLA phenotype between individuals, the immunogenic antigenic fragments (peptides) of sufficient length must contain a series of epitopes capable of being presented by several supertypes of HLA- I and -II molecules.

Also, the epitopes included in the peptide compounds of the invention have a strong immunogenic power.

T epitopes are antigenic sequences recognized by T lymphocytes. For example, in humans, T epitopes result from the degradation of antigens by presenting cells and are presented to CD8 + (cytotoxic) or CD4 + (helper) T lymphocytes by HLA molecules of class I or class II respectively. T epitopes are therefore necessarily ligands of HLA molecules and are indeed part of the peptides which bind to HLA molecules with strong or moderate affinities.

Cells expressing the major class II histocompatibility complex (MHC2) can also present microbial antigens via CD1 to gamma-delta T lymphocytes.

Presentation skills depend on many variables. They vary from one individual to another in due to the polymorphism of the major histocompatibility complex (MHC).

Thus, inbreeding, by decreasing the number of different MHCs expressed by an individual, decreases his immune capacities. They also differ according to the methods of exposure to the antigen (dose and route of administration), due to the variations in the presentation capacities of the different types of presenting cells. For example, the cells involved in the presentation will be different by the cutaneous or digestive route.

Finally, the peptide range produced by a given antigen will be different according to the presenting cell (cleavage methods), and according to the species and the individual (MHC allele).

The peptide compounds according to the invention have been selected and designed with a view to ensuring vaccination and therapeutic coverage of the populations most seriously affected by the main pathogenic species of leishmanias. They are intended to induce and characterize the prevention or treatment of conditions in mammals whose protective immunity depends on the stimulation of Thl-type lymphocytes and cytotoxic T cells, characteristic of a state of delayed hyperstimulation .

As described above, another main difficulty in the development of a vaccine candidate lies in the fact that it should ideally be effective against several species of leishmanias and in particular against the most severe clinical forms (visceral and cutaneous) and in different natural hosts of the infection (Man, dog).

The sequences of these epitopes are preferably: the peptide sequences of H2B proteins contained in Table 1 below;

the peptide sequences of LmLRAB proteins contained in Table 2 below;

the peptide sequences of PSA proteins contained in Table 3 below,

as well as their analogous derivatives, muteins and homologs.

TABLE 1: H2B

TABLE 2: LmLRAB

TABLE 3: PSA

More preferably, the sequences of these epitopes contained in the peptide compounds according to the invention are chosen from sequences SEQ ID No 1 to 21, sequences SEQ ID No 24 to 64, sequences SEQ ID No 67, 68, 70 to 72, 74 to 76, the sequences SEQ ID No 78 to 95, the sequences SEQ ID No 98 to 136, the sequences SEQ ID No 139 to 156, and the sequences SEQ ID No 158 to 184, listed in Tables 1, 2 and 3 above, as well as their analogous derivatives, muteins and homologs.

The subject of the invention is a peptide compound consisting of epitopes as described above and comprising:

(i) 1 to 21 overlapping epitopes of sequences SEQ ID No. 1 to 21 such as the sequences SEQ ID No. 22 and 23, or

(ii) 1 to 41 overlapping epitopes of sequences SEQ ID N ° 24 to 64 such as the sequences SEQ ID N ° 65 and 66, or

(iii) 1 to 18 overlapping epitopes of sequences SEQ ID No. 78 to 95 such as the sequences SEQ ID No. 96 and 97, or

(iv) 1 to 39 overlapping epitopes of sequences SEQ ID No 98 to 136 such as the sequences SEQ ID No 137 and 138, or (v) 1 to 18 overlapping epitopes of sequences SEQ ID No 139 to 156, such as the sequence SEQ ID No 157, or

(vi) 1 to 27 overlapping epitopes of sequences SEQ ID No. 158 to 184 such as the sequence SEQ ID No. 185, or

(viii) 1 to 3 epitopes of sequence SEQ ID No. 67, 68, 70 to 72, 74 to 76 such as sequences SEQ ID No. 69, 73 and 77, optionally separated by a peptide spacer or a peptide binding sequence comprising 1 to 8 amino acids.

By way of nonlimiting example of spacer or peptide binding sequence, mention may be made of the patterns ARY, KGR, RY, GR, TV, K, V, Y, L among others (Stittelaar K, et al. Vaccine, 2002 , 20: 249-261; Lee Y. et al., Biomed Microdevices, 2010, 12: 207-222; Cardinaud, S., et al. Aids, 2009, 23: 1945-1954). Preferably, the peptide linkers or sequences used are K, KGR, RY, ARY or KARY or their analogs.

More preferably, the multiepitopic peptide compounds according to the invention are chosen from the nine sequences SEQ ID No. 23, 66, 69, 73, 77, 97, 138, 157 and 185, as well as their analogous, mutein and homologous derivatives. These nine multiepitopic peptide compounds of SEQ ID N ° 23, 66, 69, 73, 77, 97, 138, 157 and 185 are for example synthetic multiepitopic peptide compounds, associated or not with peptides of the prior art having affine epitopes for HLA class I molecules.

The multiepitopic peptide compounds (24 to 40-mer), having a high affinity with a maximized number of HLA class I and class II molecules (FIGS. 4A, 4B and 4C), have a strong immunoprevalent potential corresponding to optimal vaccine coverage. (Figure 5A), in particular of the populations concerned. The coverage rates of the peptide compounds of the invention show a very high added value compared to the peptides of the prior art (Figure 5B). The in vitro efficacy of peptide compounds to stimulate human cells and produce Thl-type cytokines such as IFN-g was evaluated, in combinations to ImM for each peptide, from blood samples from cured individuals of 'a leishmaniasis, according to a short protocol and a single stimulation. As shown in the table of FIG. 6, the levels of IFN-g secreted by the stimulated T cells can reach more than 2590 pg / ml with a combination of the peptide compounds proposed and associated with peptides of the art previous, and more than 2700 pg / mL with a combination of the proposed peptide compounds not associated with these peptides. These rates show a very high added value compared to the peptides of the prior art corresponding to "Pool Z" in the table in FIG. 6. In this table, "Cured CL 1 to 6" are individuals cured of cutaneous leishmaniasis , "Naive 1 to 6" are naive individuals (non-immune control), "NS" corresponds to "unstimulated" (medium alone) after 5 ("NS 5 days") and 10 days ("NS 10 days") of culture, "PHA" is Phytohaemagglutinin (positive stimulation control), "SLA" corresponds to the total soluble antigens of Leishmania major, and "Pool A to G" correspond to combinations of the synthetic peptide compounds of sequence SEQ ID No 23, 66, 69, 73, 77, 97, 138, 157 and 185, associated (Pool A, B, C, D, F) or not (Pool E, G) with synthetic peptide compounds of the prior art. The values of IFN-g for "PHA", "SLA" are the values subtracted from the values "NS 5 days", the values of IFN-y for the "Pool A to Z" are the values subtracted from the values of " NS 10 days "and the values in bold correspond to values higher than the Average of Naive + 3 x standard deviation. The in vitro immunogenicity of peptide compounds is the capacity of peptides to recruit precursor lymphocytes, to induce in vitro stimulation of specific T lymphocytes with production of IFN-g. The immunogenicity was evaluated, with combinations of peptides (combination corresponding to "Pool H" in FIG. 7) at ImM for each peptide, from blood samples from naive individuals and according to a long protocol with at least three successive stimulation. An IFN-g ELISPOT test to measure the frequency of preexisting naive T cells and the amplitude of the naive T repertoire specific for each peptide, was performed with CD4 + T cells and autologous dendritic cells of a naive individual in presence or absence of the mixture of peptides, or in the presence of each peptide individually. Several lines specific to the peptide combination have been identified and respond specifically to at least one of the peptides of the present invention (Figures 8 and 9). The combination "Pool H" was used either with unmodified peptides at the amino-terminal end and with amidation of the carboxy-terminal end (FIG. 8), or with peptides chemically modified by a palmitoyl tail at the amino-terminal end and with amidation of the carboxy-terminal end (Figure 9).

The invention further relates to a composition as a pharmaceutical product, for human or veterinary use comprising at least:

an epitope of sequence chosen from sequences SEQ ID No 1 to 21, sequences SEQ ID No 24 to 64, sequences SEQ ID No 67, 68, 70 to 72, 74 to 76, sequences SEQ ID No 78 to 95, the sequences SEQ ID No 98 to 136, the sequences SEQ ID No 139 to 156, and the sequences SEQ ID No 158 to 184, or a peptide compound comprising 1, 2, 3 or 4 of the epitopes of sequence SEQ ID No 1 to 21, SEQ ID No 24 to 64, SEQ ID No 67, 68, 70 to 72, 74 to 76, SEQ ID No 78 to 95 , SEQ ID No 98 to 136, SEQ ID No 139 to 156, and SEQ ID No 158 to 184, optionally separated by a spacer as defined above, or

a peptide compound of sequence chosen from SEQ ID No 22, 23, 65, 66, 69, 73, 77, 96, 97, 137, 138, 157 and 185.

The invention also relates to such a composition for its use in prophylactic and therapeutic vaccination directed against one or more of the leishmanias such as Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, Leishmania (Viannia) peruviana.

Advantageously, the composition according to the invention is suitable for use in prophylactic and therapeutic vaccination directed against at least 3, preferably at least 7, more preferably at least 10 and more preferably against all of the leishmanias listed above. -above.

The composition advantageously comprises an adjuvant chosen from adjuvants chosen from Toll-Like Receptor adjuvants of the TLR3, TLR4, TLR5, TLR7, TLR8, TLR9 classes, saponins and their derivatives QA21, quilA or QS21, oil in water or water in oil emulsions, polysaccharides, cationic liposomes, virosomes or polyelectrolytes and immunomodulators chosen from sandfly saliva proteins, cytokines, peptides and heat shock proteins, for example HSP70.

Preferably, the composition according to the invention is administered by subcutaneous, intradermal, intramuscular, intravenous, parenteral, endonasal, mucosal or oral route, and is therefore presented in a form suitable for such administrations.

An additional subject of the invention relates to a composition as defined above as a medicament, vaccine, or reagent for diagnostic in vitro and / or in vivo, for the induction or diagnosis, in a mammal, of transition from a Th2 type immune state to a Th1 type immune state.

A further additional object of the invention relates to a vaccine capable of imparting cross-immunoprotection against the various species of leishmaniasis. 1 / large antigenic community shared by the leishmanias makes it possible to envisage the development of a single polyvalent vaccine, consisting of common and highly conserved immunogens. Targeting a common antigen (s) for all leishmania species as a vaccine should no doubt represent a real advantage in terms of cross-vaccination. Such a vaccine includes at least:

an epitope of sequence chosen from sequences SEQ ID No 1 to 21, sequences SEQ ID No 24 to 64, sequences SEQ ID No 67, 68, 70 to 72, 74 to 76, sequences SEQ ID No 78 to 95, SEQ ID No 98 to 136, the sequences SEQ ID No 139 to 156, and the sequences SEQ ID No 158 to 184, or

a peptide compound comprising 1, 2, 3 or 4 of the epitopes of sequence SEQ ID No 1 to 21, SEQ ID No 24 to 64, SEQ ID No 67, 68, 70 to 72, 74 to 76, SEQ ID No 78 to 95 , SEQ ID No 98 to 136, SEQ ID No 139 to 156, and SEQ ID No 158 to 184, optionally separated by a spacer as defined above, or

Such a vaccine is advantageously used for a prophylactic and therapeutic vaccination directed against one or more of the leishmanias chosen from Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania Tropica, Leishmania aiop, Leishmania aiop Viannia) hraziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, Leishmania (Viannia) peruvian.

The vaccine which is the subject of the invention is advantageously intended for humans, canids, felines and equines. Preferably, the vaccine which is the subject of the invention is intended for humans and dogs.

Claims

1. Peptide compound comprising at least 2 epitopes contained in the protein sequences chosen from the PSA, H2B or LmLRAB proteins of Leishmania having a sequence chosen from the sequences SEQ ID No 1 to 21, the sequences SEQ ID No 24 to 64, sequences SEQ ID No 67, 68, 70 to 72, 74 to 76, sequences SEQ ID No 78 to 95, sequences SEQ ID No 98 to 136, sequences SEQ ID No 139 to 156, and sequences SEQ ID No 158 to 184, as well as their analogous derivatives, muteins and homologs exhibiting immunogenic power, said epitopes being optionally separated by a peptide spacer comprising at least 1 amino acid.

2. Peptide compound according to claim 1, in which said analogous derivatives, muteins and homologs of the epitopes, having an immunogenic power, have a percentage of sequence identity of at least 50%, preferably at least 75% with the sequence of said epitopes.

3. Peptide compound according to one of claims 1 or 2, characterized in that the peptide spacer comprises 1 to 8 amino acids.

4. Peptide compound according to one of claims 1, 2 or 3, characterized in that it comprises 2, 3 or 4 of said epitopes.

5. Peptide compound according to one of the preceding claims, in which a first epitope is chosen from said epitopes contained in the sequence of a first protein chosen from PSA, H2B or LmLRAB, and a second epitope is contained in the sequence of a second protein, different from the first protein, chosen from PSA, H2B or LmLRAB.

6. Peptide compound according to one of the preceding claims, characterized in that it is linked to a carrier group which increases its immunogenic character.

7. Peptide compound according to one of the preceding claims, in which the epitope (s) are chosen from the epitopes of the sequences SEQ ID No 22, 23, 65, 66, 69, 73, 77, 96, 97, 137, 138, 157 and 185.

8. Peptide compound according to one of the preceding claims, characterized in that it comprises at least 3 epitopes contained in the sequences of different proteins chosen from PSA, H2B and LmLRAB.

9. Specific antibody and immuniserum containing it directed against epitopes of peptide compounds according to one of the preceding claims.

10. Pharmaceutical composition comprising at least one peptide compound according to one of claims 1 to

11. Composition comprising at least one peptide compound according to one of claims 1 to 8, for its use in prophylactic and therapeutic vaccination directed against leishmanias.

12. Composition for its use according to claim 11, characterized in that the leishmanias are Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania (Viannia) brazlllensls, Leishmania (Viannia) guyanensls, Leishmania (Viannia) panamensis and / or Leishmania (Viannia) peruviana.

13. Composition comprising at least one peptide compound according to one of claims 1 to 8, for the manufacture of a medicament or of a vaccine, of an in vivo or in vitro diagnostic reagent for induction or diagnosis in mammals, activation of cell-mediated immunity dependent on Thl-type lymphocytes and / or effector humoral immunity.

14. Prophylactic and / or therapeutic vaccine for its use against one or more of the leishmanias chosen from Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania majoria Leishmania aethiopica, braziliensis, Leishmania (Viannia) guyanensls, Leishmania (Viannia) panamensis, and / or Leishmania (Viannia) peruviana, comprising at least one peptide compound according to one of claims 1 to 8.

15. Vaccine for its use according to claim 14, characterized in that it comprises on the one hand, at least one peptide compound chosen from SEQ ID No. 22, 23, 69, 73 and 77, 65, 66, 96, 97, 137, 138, 157 and 185, as well as its analogous derivatives, muteins and homologs.

16. Vaccine for its use according to one of claims 14 or 15, characterized in that it comprises on the one hand, at least one or two peptide compounds chosen from SEQ ID No. 22, 23, 69, 73 and 77 as well as its analogous, mutein and homologous derivatives; and on the other hand, at least one, two or three peptide compounds chosen from the sequences SEQ ID No. 65, 66, 96, 97, 137, 138, 157 and 185 as well as its analogous, mutein and homologous derivatives.

17. Vaccine for its use according to one of claims 14 to 16, characterized in that it comprises, in combination, the following multiepitopic peptide compounds:

SEQ ID No 23, 66, 73, 77, 97, 138, 157 and 185 as well as its analogous derivatives, muteins and homologs; or

SEQ ID No 23, 66, 157 and 185 as well as its analogous derivatives, muteins and homologs;

SEQ ID No 66, 97, 138, 157 and 185 as well as its analogous derivatives, muteins and homologs; or

SEQ ID No 66, 157 and 185 as well as its analogous derivatives, muteins and homologs.

18. Vaccine for its use according to one of claims 14 to 17, characterized in that it further comprises an adjuvant chosen from adjuvants of classes TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, saponins and their derivatives QA21, quilA or QS21, oil in water or water in oil emulsions, polysaccharides, cationic liposomes, virosomes or polyelectrolytes and immunomodulators chosen from sandfly saliva proteins, cytokines, peptides and proteins thermal shock.

19. Vaccine for its use according to one of claims 14 to 18, for sub-administration cutaneous, intradermal, intramuscular, parenteral, endonasal, mucosal or oral.

20. Nucleotide sequence coding for the peptide compounds defined in one of claims 1 to 8, or the epitopes contained in these peptide compounds.

21. Expression vector comprising at least one nucleotide sequence as defined in claim 20, as well as the means necessary for their expression.

22. Diagnostic reagent comprising a peptide compound according to one of claims 1 to 8.