Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/008—Leishmania antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55583—Polysaccharides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to 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.
• LC cutaneous-mucous
• LV visceral cutaneous
• leishmaniasis must take into account asymptomatic individuals, who are considered as potential reservoirs of leishmaniases.
• Vaccination is the most suitable way to interrupt the transmission of Leishmania parasites and eliminate leishmaniasis.
• canine vaccines are used. Examples include the CaniLeish TM, Leish-Tec TM or Leishmune TM vaccines. However, no vaccine for human application is available.
• Such a human vaccine would, however, have significant health and socio-economic consequences.
• 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.
• 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.
• PBMC peripheral blood
• 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.
• 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,
• 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
• 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.
• 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;
• 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 °
• 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,
• 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,
• 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
• 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).
• 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.
• the MPL10-12 line responds to peptides SEQ ID No 23
• 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).
• MPL10 naive individual
• PAL-peptide-NH2 an NH2 group at the carboxy-terminal end
• 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.
• epitope means a peptide compound or peptide defined by its sequence having approximately 8 to 15 amino acids.
• 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 .
• 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.
• 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.
• 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.
• the term “homologous derivatives” means peptide compounds having a certain percentage of peptide identity.
• 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.
• 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.
• MHC Major Histocompatibility Complex
• 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:
• 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;
• 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
• 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.
• 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.
• the epitopes or peptide compounds which are the subject of the invention are linked to carriers making it possible to make them more immunogenic.
• carriers include carrier proteins KLM (Keyhole limpet hemocyanin), lipopeptides of palmitoyl type, or their derivatives.
• KLM Keyhole limpet hemocyanin
• 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.
• 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.
• 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.
• 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.
• pseudo-peptide bond is understood to mean all types of bonds capable of replacing the conventional peptide bonds.
• the geometry of the molecules is such that they can theoretically be in the form of different optical isomers.
• 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)
• 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.
• chemical synthesis in the laboratory makes it possible to prepare amino acids having the two possible conformations.
• 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.
• MHC Major Complex Histocompatibility
• HLA human leukocyte antigens
• 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.
• 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.
• 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 can also present microbial antigens via CD1 to gamma-delta T lymphocytes.
• MHC major histocompatibility complex
• 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.
• 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 .
• sequences of these epitopes are preferably: the peptide sequences of H2B proteins contained in Table 1 below;
• 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:
• spacer or peptide binding sequence 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).
• the peptide linkers or sequences used are K, KGR, RY, ARY or KARY or their analogs.
• 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.
• 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.
• 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.
• the invention further relates to a composition as a pharmaceutical product, for human or veterinary use comprising at least:
• 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.
• leishmanias such as Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania (Viannia) braziliens
• 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.
• 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.
• 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 s
• 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.
• a vaccine includes at least:
• 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.
• leishmanias chosen from Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania Tropica, Leishmania aiop, Leishmania aiop Viannia) hraziliensis, Leishmania (Vianni
• the vaccine which is the subject of the invention is advantageously intended for humans, canids, felines and equines.
• the vaccine which is the subject of the invention is intended for humans and dogs.

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• 2018
  • 2018-05-30 FR FR1800533A patent/FR3081870B1/fr active Active
• 2019
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