WO2020245433A1 - Mixtures of immunogenic cd8 t epitopes of the ebola virus - Google Patents

Mixtures of immunogenic cd8 t epitopes of the ebola virus Download PDF

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WO2020245433A1
WO2020245433A1 PCT/EP2020/065733 EP2020065733W WO2020245433A1 WO 2020245433 A1 WO2020245433 A1 WO 2020245433A1 EP 2020065733 W EP2020065733 W EP 2020065733W WO 2020245433 A1 WO2020245433 A1 WO 2020245433A1
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hla
seq
restricted
epitopes
epitope
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French (fr)
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Bernard Maillere
Yann GALLAIS
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Commissariat A L'energie Atomique Et Aux Energies Alternatives
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising a combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules and of inducing a lymphocyte response Human CD8 T specific in individuals expressing at least one of said HLA I molecules.
  • the invention also relates to the use of said composition as a preventive vaccine against Ebola virus and as a reagent for immunomonitoring the cellular response against the virus. Ebola.
  • the Ebola virus is a highly virulent virus of the Filoviridae family responsible for epidemics of fatal hemorrhagic fevers in humans and non-human primates.
  • This virus comprises a single envelope protein, Glycoprotein (GP), as well as internal proteins including Nucleoprotein (NP) which is the most expressed viral protein with around 3200 molecules per virion and potentially the most immunogenic.
  • Glycoprotein Glycoprotein
  • NP Nucleoprotein
  • Five species of the Ebola virus have been identified: Zaire, Sudan, Reston, Forêt de Ta ⁇ and Bundibugyo.
  • Zaire Ebola virus (EBOV) has been responsible for most epidemic outbreaks since the discovery of Ebola virus in 1976, with an average case fatality rate of around 50% (25% to 90% in recent outbreaks).
  • GP glycoprotein
  • replicative viral vectors such as vesicular stomatitis virus (rVSV-Ebov), cytomegalovirus (rCMV), or human parainfluenza virus type 3 (HPIV3), as well as non-replicating adenoviruses such as Chad3-EBOV (Chimp adenovirus), EBOLA rAd-5 and Ad26.ZEBOV.
  • rVSV-Ebov vesicular stomatitis virus
  • rCMV cytomegalovirus
  • HPIV3 human parainfluenza virus type 3
  • non-replicating adenoviruses such as Chad3-EBOV (Chimp adenovirus), EBOLA rAd-5 and Ad26.ZEBOV.
  • plasmids INO-4212 or VRC-EBODNA023-00-VP
  • nanoparticles containing the recombinant glycoprotein (GP) combined with a saponin-based adjuvant Matrix-M TM.
  • CD8 + T lymphocytes play a major role in the immune response against the Ebola virus.
  • a correlation between survival to infection and the level of activated CD8 + T lymphocytes has in fact been shown (Sanchez et al., J. Virol., 2004, 78, 10370-10377). It has also been shown that mice vaccinated with the Venezuelan Equine Encephalitis Virus encoding Ebola PN developed a protein-specific CD8 + response, thereby protecting the animals from lethal infection (Wilson and Hart, J. Virol., 2001, 75, 2660-2664). In contrast, little data is available on the role of a cellular response against GP.
  • Cytotoxic CD8 + T lymphocytes are capable of recognizing the antigens of a pathogenic virus, in the form of peptides with a length of 9-10 amino acids, called CD8 + T epitopes (or CD8 T epitope), presented by molecules of the Class I Major Histocompatibility Complex (HLA I (HLA-A, HLA-B and HLA-C) in humans) on the surface of infected cells and lyse the infected cells directly after recognition of the peptides. Due to the polymorphism of HLA I molecules in the population, the binding specificity of HLA I molecules is highly variable from one HLA class I molecule to another.
  • HLA I Class I Major Histocompatibility Complex
  • HLA I alleles All of the HLA I alleles of an individual is referred to as HLA I typing. Consequently, in order to develop an effective vaccine, it is essential that the latter contain CD8 + T epitopes which can be presented by different HLA I molecules in order to have the widest possible vaccination coverage.
  • the inventors have identified numerous CD8 T epitopes of NP and GP of the Zaire Ebola virus capable of stimulating specific CD8 T lymphocytes in a panel of healthy donors representative of Caucasian and African populations ([Table 2], [ Table 3] and [Table 4]; Figure 1).
  • CD8 T epitopes which are immunogenic in humans which are restricted to only 3 predominant HLA I molecules (HLA-A2, HLA-A23, HLA-B35)
  • these CD8 T epitopes are immunogenic in human individuals carrying various HLA I molecules including all of the most frequent HLA-A and HLA-B molecules in Caucasian and African populations (HLA-A1, -A2, - A3, -A11, -A24, -A29, -B7, - B8, -B15, -B18, -B35, -B40, -B44 and -B51) with the exception of HLA-A23 ([Table 1]) .
  • HLA-A and HLA-B alleles make it possible to '' achieve vaccination coverage of potentially 95.2% of individuals in the world population; 98.7% of people in Europe; between 67.9 and 80.9% of people in Africa; between 78.8% and 92.2% of individuals from Asia and Oceania; and between 73.4% to 95.1% of people in America and the Caribbean.
  • PN is more immunogenic than GP, 54 epitopes having been obtained for the first and 28 for the second, in agreement with previous observations in patients who have been infected with the virus (McElroy 2015, supra).
  • the NP sequence is more conserved and the CD8 T epitopes derived from NP are therefore potentially effective against the various strains of Ebola.
  • CD8 T epitopes of the invention By combining several CD8 T epitopes of the invention, and optionally CD4 T epitopes, in one or more peptides / polypeptides derived from the NP or the GP of the Ebola virus, playpitope polypeptides or chimeric proteins, and / or polynucleotides, recombinant vectors , we thus obtain a vaccine against the Ebola virus which has an optimal potential vaccination coverage in the various populations, in particular African and Caucasian. Eleven NP epitopes and 10 GP epitopes allow maximum vaccination coverage in the reference population (100% of responder individuals corresponding to 92% and 60% of individuals in the population, respectively for the peptides of the
  • NP and GP peptides By combining several T CD8 and T CD4 epitopes in long polypeptide fragments (LSP), 6 LSPs for both NP and GP, we obtain a maximum potential vaccination coverage of the reference population, for the responses in CD8 T lymphocytes and CD4 T lymphocytes specific for the Ebola virus ([Table 12] and [Table 13]).
  • LSP long polypeptide fragments
  • the present invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising at least one combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules chosen from HLA -A1, HLA-A2, HLA-A3, HLA- A11, HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA- B40, HLA-B44 and HLA-B51 and inducing a specific human CD8 + T lymphocyte response in individuals expressing at least one of said HLA-I molecules, and said epitopes being selected from the group consisting of:
  • said immunogenic or vaccine composition comprises a combination of epitopes chosen so that it is suitable for being presented by at least 8 different predominant HLA I molecules, preferably at least 12, 13 or all of said predominant HLA I molecules.
  • said composition comprises a combination of epitopes selected from the group consisting of: the sequences SEQ ID NO: 3 to 7, 9 to 22, 24 to 35 and 37 to 48 of the NP and the sequences SEQ ID NO: 50 to 52, 54 to 56 and 59 to 64 and 66 to 71 of the GP.
  • the immunogenic or vaccine composition comprises at least one epitope selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 15, 17, 24, 25, 34, 41 and 42 of the NP and the sequences SEQ ID NO: 55, 61 and 71 of the GP.
  • said immunogenic or vaccine composition comprises at least one CD8 T epitope conserved in different strains of the Ebola virus, chosen from the sequences SEQ ID NO: 3 to 8, 10 to 39, 41, 43, 47 and 48 of the NP and the sequences SEQ ID NO: 51 to 60 and 67 to 71 of the GP; preferably chosen from the sequences SEQ ID NO: 5, 6, 12, 15 to 24, 27 to 38 and 41 of the NP and the sequences SEQ ID NO: 51, 54, 55, 57 to 60, 67 and 70 of the GP; even more preferably among the sequences SEQ ID NO: SEQ ID NO: 15 to 18, 20 to 24, 27, 30 to 33, 36 and 37 of the NP and the sequences SEQ ID NO: 55, 57, 58, 60 and 67 of the GP.
  • said immunogenic or vaccine composition comprises a combination of CD8 T epitopes from NP or a combination of CD8 T epitopes from NP and GP.
  • said immunogenic or vaccine composition comprises a combination of CD8 T epitopes of NP comprising at least:
  • epitope SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15 or one of the epitopes SEQ ID NO: 10 or SEQ ID NO: 41 restricted to HLA-A29; preferably the epitope SEQ ID NO: 6;
  • said immunogenic or vaccine composition comprises a combination of CD8 T epitopes of GP comprising at least
  • said immunogenic or vaccine composition further comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of the GP.
  • said combination of CD8 T epitopes, and optionally the other epitopes are included in one or more peptides derived from the NP protein of sequence SEQ ID NO: 1 or GP of sequence SEQ ID NO: 49, multi-epitope polypeptides or chimeric proteins, and / or polynucleotides or recombinant vectors encoding said peptide (s), polypeptides and / or proteins.
  • said immunogenic or vaccine composition comprises the NP peptides of sequence SEQ ID NO: 80, 135, 149, 150, 151 and 152 or the GP peptides of sequence SEQ ID NO: 107, 148, 153, 154, 155 and 156 .
  • a subject of the present invention is also said immunogenic or vaccine composition according to the present description for its use as a vaccine in the prevention of an infection by the Ebola virus.
  • a subject of the present invention is also the in vitro use of said immunogenic or vaccine composition according to the present description for the diagnosis of an infection by the Ebola virus or the immunomonitoring of the cellular response against the Ebola virus. Disclosure of the invention
  • the present invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising at least one combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules. and inducing a human CD8 + T lymphocyte response specific for Ebola virus in individuals expressing at least one of said HLA-I molecules.
  • sequences derived from the nucleoprotein (NP) of the Ebola virus are defined with reference to the NP of sequence SEQ ID NO: 1 or UniProt
  • AAD14590.1 from the Zaire strain of the Ebola virus (ZEBOV).
  • sequences derived from the glycoprotein (GP) of the Ebola virus are defined with reference to the GP of sequence SEQ ID NO: 49 or UniProt AKG65268.1, derived from the Zaire strain of the Ebola virus (ZEBOV).
  • CD8 T epitope means a peptide of 9 to 10 amino acids which binds at least one HLA I molecule, preferably a predominant HLA I molecule as presented in [Table 1], and is thus presented by said HLA I molecule. and recognized by specific human CD8 T lymphocytes in individuals carrying this HLA I molecule.
  • CD8 T epitope restricted to an HLA I molecule is understood to mean an epitope which is recognized by specific human CD8 T lymphocytes when it is presented by said HLA I molecule.
  • immunological CD8 T epitope means a CD8 T epitope which is capable of inducing a response in specific human CD8 T lymphocytes in individuals expressing at least said HLA I molecule.
  • - The term “predominant HLA I molecule” means an HLA I molecule whose allelic frequency is greater than 5% in a reference population, for example one of the Caucasian or African populations of [Table 1].
  • - CD4 T epitope is understood to mean a peptide of 11 to 25 amino acids which binds at least one HLA II molecule, in particular at least one HLA-DR allele frequent in different populations as presented in [Table 7], and is recognized by specific human CD4 T lymphocytes in individuals carrying this HLA II molecule; the peptide comprises a sequence of 9 amino acids including the residues for anchoring to the HLA II molecules, flanked by at least 1 amino acid, preferably at least 2 or 3 amino acids at each of its ends.
  • specific CD8 T lymphocytes or CD4 T lymphocytes means CD8 T lymphocytes or CD4 T lymphocytes specific for the NP or GP sequences of the Ebola virus.
  • the expressions “specific CD8 or CD4 T lymphocytes”, “CD8 T lymphocytes specific for the Ebola virus”, “CD8 T lymphocytes specific for the NP or the GP of the Ebola virus”, “CD8 T lymphocytes directed against the Ebola virus”, “CD8 T lymphocytes directed against the NP or GP Ebola virus” are used interchangeably to designate CD8 T lymphocytes or CD4 T lymphocytes specific for the sequences of the NP or the GP of the Ebola virus.
  • induction of a response in specific CD8 T lymphocytes or CD4 T lymphocytes means the stimulation of specific CD8 T lymphocytes or CD4 T lymphocytes in vitro or in vivo.
  • frequency or level of responders in vitro to a T CD4 or T CD8 epitope or a peptide comprising a T CD4 or T CD8 epitope according to the present invention is understood to mean the percentage of human individuals of a reference group for which the lines human CD4 or CD8 T lymphocytes specific for said epitope or peptide were obtained.
  • the term “intensity of the response in vitro to a peptide comprising a CD4 T epitope according to the present invention” means the percentage of CD4 T lymphocyte lines specific for said peptide which were obtained in a group of reference human individuals compared to all of the specific CD4 T lymphocyte lines obtained with the different peptides of the same protein (NP or GP).
  • the term "reference group” is understood to mean a set of human individuals expressing various HLA I or HLA II molecules, in particular various HLA I molecules including the most frequent HLA-A and HLA-B alleles in different populations ([Table 1] or various HLA-DR molecules including the most frequent HLA-DR alleles in different populations ([Table 7]).
  • the reference group includes the reference groups of the examples (HLA I: Figure 1, [Table 2], [Table 3] and [Table 4]); HLA II: [Table 8] and [Table 9]).
  • the reference group is considered to be representative of a population or a set of populations, that is to say it provides results that can be extrapolated to this or these population (s).
  • Ebola virus is understood to mean any isolate of Ebola virus.
  • the percentage identity of an amino acid sequence is defined by the percentage of amino acid residues in a sequence to be compared which are identical to a reference sequence after alignment of the sequences, by introducing spaces if necessary, so as to obtain maximum sequence identity.
  • the alignment of sequences with a view to determining the percentage identity of a sequence can be carried out in various ways known to those skilled in the art, for example using publicly available software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). This software is preferably used with default settings.
  • a means at least one, and or means and / or.
  • CD8 T epitopes and derivative peptides comprising these epitopes according to the invention to bind HLA I molecules is evaluated according to standard techniques known to those skilled in the art such as those described in particular in the examples. These are in particular in silico methods The in silico methods use tools for predicting binding to MHC-I, such as for example NetMHC v4.0.
  • CD8 T epitopes and peptides derived from the invention to stimulate specific human CD8 T lymphocytes, for example from precursors present in naive human individuals, to specifically stimulate such cells in human individuals who have been infected with the Ebola virus, the specificity of the CD8 T lymphocytes induced vis-à-vis the peptides or the NP or GP protein of the Ebola virus, as well as the ability of the CD8 T epitopes and peptides derived according to the invention to be recognized by lymphocytes T CD8 specific, is evaluated according to standard techniques known to those skilled in the art such as those described in the examples.
  • a cell proliferation test an ELISPOT test (assay of cytokine-producing cells) or an assay for assaying intracellular cytokines, specific for a cytokine produced by activated CD8 T lymphocytes, in in particular a Th1 type cytokine such as, for example, IFN-g, IL-2 or TNF-a.
  • a Th1 type cytokine such as, for example, IFN-g, IL-2 or TNF-a.
  • the examples show that the CD8 T epitopes and peptides derived according to the invention are capable of inducing a response in specific human CD8 T lymphocytes from precursors present in naive human individuals of a reference group.
  • said predominant HLA I molecules are chosen from HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A29, HLA-B7, HLA -B8, HLA-B15, HLA-B18, HLA-B35, HLA-B40, HLA-B44 and HLA-B51; preferably from HLA-A * 01: 01, HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11: 01, HLA-A * 24: 02, HLA-A * 29: 02 , HLA-B * 07: 02, HLA-B * 08: 01, HLA-
  • this set of alleles covers 95.2% of individuals in the world population; 98.7% of people in Europe; 67.9% of people from Central Africa; 71.9 individuals from East Africa; 80.8 people from Sub-Saharan Africa; 80.7 of individuals from North Africa and 74.4% of individuals from West Africa; 87.9% of individuals from Northeast Asia; 78.8% of individuals from South Asia; 88.9% of individuals from Southeast Asia; 81.2% of individuals from Southwest Asia; 92.2% of individuals from East Asia; 92.1% of people from Oceania; 95.1% of people from North America; 73.4% of people from South America and 93.4% of people from the Caribbean
  • said CD8 epitopes are selected from the following sequences of the nucleoprotein (NP) and / or of the glycoprotein (GP) of the Ebola virus: - SEQ ID NO: 28 and 46 of the NP, restricted to HLA-A1
  • the above sequences are from the Zaire Ebola virus.
  • the invention encompasses the natural or synthetic variant epitopes obtained by mutation (insertion, deletion, substitution) of one or more amino acids in the sequence of the nucleoprotein of sequence SEQ ID NO: 1 or of the glycoprotein of sequence SEQ ID NO: 49, since said peptide retains its properties of the immunogenic CD8 T epitope of the Ebola virus as defined above, it is that is, it is recognized by human CD8 T lymphocytes specific for the wild-type sequence or for a natural variant of the Ebola virus.
  • the variant epitopes advantageously comprise at most 3 mutations in one of the sequences SEQ ID NO: 2 to 48 and 50 to 73, preferably at most 3 substitutions in said sequences.
  • Said combination of epitopes advantageously comprises 5 or more CD8 T epitopes according to the invention, for example 5 to 15 (5, 6, 7, 8, 9, 10, 1 1, 12, 13,
  • the combination of epitopes of the composition is chosen so that it is capable of being presented by at least 4 different predominant HLA I molecules, preferably at least 8 (8, 9, 10, 11, 12, 13 or 14) different preponderant HLA I molecules, preferably at least 12 (12, 13 or 14) different preponderant HLA I molecules, even more preferably 13 or all of said preponderant HLA I molecules.
  • composition comprising a combination of CD8 T epitopes of the NP and / or of the GP of the Ebola virus as defined above makes it possible to improve the immune response against the Ebola virus by broadening the coverage of responder human individuals.
  • said combination of epitopes comprises at least one epitope selected from the group consisting of the sequences SEQ ID NO: 6, 21, 27, 34, 35, 42 of the NP and GP SEQ ID NO: 54, 55 and 61; these epitopes are restricted to several HLA I molecules, thus making it possible to limit the number of peptide sequences in the composition.
  • the epitopes of the combination are selected from the group consisting of: the sequences SEQ ID NO: 3 to 7, 9 to 22, 24 to 35 and 37 to 48 of the NP and the sequences SEQ ID NO: 50 to 52, 54 to 56 and 59 to 64 and 66 to 71 of the GP.
  • These epitopes advantageously induce a responder frequency of at least 25% in individuals carrying the allele or one of the HLA I alleles to which the epitope is restricted as defined above ([Table 2], [Table 3 ] and [Table 4]).
  • At least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5 to 7, 10 to 21 (HLA-A2), 22, 24 to 35, 37 to 45 and 48 of the NP and the sequences SEQ ID NO: 52, 54 (HLA-B40), 55, 56, 59, 61 (HLA-A29), 63, 64, 66 and 68 to 71 of the GP.
  • These epitopes advantageously induce a responder frequency of at least 33.3% in individuals carrying the allele or one of the HLA I alleles to which the epitope is restricted as defined above or the HLA I allele such as as indicated in parentheses ([Table 2], [Table 3] and [Table 4]).
  • At least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 12, 14 to 17, 20, 22, 24 to 27 (HLA-B7), 28 to 31, 33, 34, 35 (HLA-B35), 37, 39 to 42, 44, 45 of the NP and the sequences SEQ ID NO: 52, 54 (HLA-B40), 55 (HLA-B18), 59, 61 (HLA-A29), 63, 64, 66, 68, 69 and 71 of the GP.
  • These epitopes advantageously induce a responder frequency of at least 50% in individuals carrying the allele or one of the HLA I alleles as defined above or the HLA I allele as indicated in parentheses ([Table 2], [Table 3] and [Table 4]) ⁇
  • At least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 15, 17, 24, 25, 34, 41 and 42 of NP and the sequences SEQ ID NO: 55 (HLA-B18), 61 (HLA-A29) and 71 of GP.
  • These epitopes advantageously induce a responder frequency of 75% to 100% in individuals carrying the allele or one of the HLA I alleles as defined above or the HLA I allele as indicated in parentheses ([Table 2], [Table 3] and [Table 4]) ⁇
  • said combination comprises at least one conserved CD8 T epitope, that is to say the sequence of which is conserved in at least one other species of the Ebola virus, of preference Ebola Sudan; preferably a sequence conserved in Ebola Zaire Sudan, Reston, Bundibugyo and Tai Forest.
  • the sequence of said peptide has at least 66%, preferably at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the sequence of another Ebola virus species, preferably Ebola Sudan; preferably with the sequences of the Ebola Sudan, Reston, Bundibugyo and Tai Forest viruses.
  • the composition advantageously comprises at least one NP peptide chosen from the sequences SEQ ID NO: 3 to 8, 10 to 39, 41, 43, 47 and 48 which have at least 66% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 5, 6, 12, 15 to 24, 27 to 38 and 41 which have at least 85% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 15 to 18, 20 to 24, 27, 30 to 33, 36 and 37 which have at least 90% identity with the sequences of Ebola Sudan, Reston, Bundibugyo and Tai Forest or at least one GP peptide chosen from the sequences SEQ ID NO: 51 to 60 and 67 to 71 which have at least 66% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 51, 54, 55, 57 to 60, 67 and 70 which have at least 85% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 55, 57, 58, 60
  • said combination comprises CD8 T epitopes from NP, CD8 T epitopes from GP or a mixture of TCD8 epitopes from NP and GP.
  • said combination comprises CD8 T epitopes of NP or a mixture of TCD8 epitopes of NP and GP.
  • said composition comprises a combination of CD8 T epitopes of the NP of the Ebola virus which is capable of being presented by all of said HLA I molecules and of inducing a maximum frequency of responders in vitro, that is to say in all the individuals of a reference group who are responders to PN (corresponding to 92% of the individuals in the reference group of the examples; see [Table 2] and [Table 3]), said combination comprising
  • epitope SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15 or one of the epitopes SEQ ID NO: 10 or SEQ ID NO: 41 restricted to HLA-A29; preferably the epitope SEQ ID NO: 6;
  • SEQ ID NO: 42 restricted to HLA-B40 and HLA-B18, preferably the epitope SEQ ID NO: 30, SEQ ID NO: 34 or SEQ ID NO: 42, more preferably the epitope SEQ ID NO: 34 or SEQ ID NO: 42 and;
  • HLA-B35 preferably the epitope SEQ ID NO: 27 or SEQ ID NO: 35; more preferably the epitope SEQ ID NO: 27;
  • said composition comprises at least the epitopes SEQ ID NO: 5 restricted to HLA-A2; SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 17 restricted to HLA-A2, SEQ ID NO: 24 restricted to HLA-A1 1, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 27 restricted to HLA-B7 and HLA-B51, SEQ ID NO: 28 restricted to HLA-A1, SEQ ID NO: 31 restricted to HLA-B44, SEQ ID NO : 34 or SEQ ID NO: 42 restricted to HLA-B40 and HLA-B18 and SEQ ID NO: 44 restricted to HLA-A3.
  • said composition comprises at least the epitopes SEQ ID NO: 5, SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 9 restricted to HLA-A3, SEQ ID NO: 10 restricted to HLA-A29, SEQ ID NO: 15 restricted to HLA-B15, SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 17 restricted to HLA-A2, SEQ ID NO: 18 restricted to HLA-B51, SEQ ID NO: 20 restricted to HLA-A2, SEQ ID NO: 21 restricted to HLA-A2 and HLA-A24, SEQ ID NO: 22 restricted to HLA -B15, SEQ ID NO: 24 restricted to HLA-A1 1, SEQ ID NO: 25 restricted to HLA-B18, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 27 restricted to HLA-B7 and HLA- B51, SEQ ID NO:
  • composition comprising said combination of CD8 T epitopes of Ebola virus NP is capable of being presented by all of said HLA I molecules and of inducing a maximum frequency of responders in vitro, that is to say in all individuals. of a reference group who are responders to PN (corresponding to 92% of individuals in the reference group of the examples; see [Table 12]).
  • said composition comprises a combination of CD8 T epitopes of the Ebola virus GP which is capable of being presented by 13 of said predominant HLA I molecules (all HLA I molecules selected with the exception of HLA-B8) and to induce a maximum frequency of responders in vitro, that is to say in all the individuals of a reference group who are responders to GP (corresponding to 60% of individuals in the reference group of the examples; see [Table 4]), said combination comprising at least the following epitopes:
  • said composition comprises at least the epitopes SEQ ID NO: 51 restricted to HLA-A24; SEQ ID NO: 52 restricted to HLA-B15, SEQ ID NO: 53 restricted to HLA-A2, SEQ ID NO: 54 restricted to HLA-B40 and HLA-B44, SEQ ID NO: 55 restricted to HLA-B7, HLA- B18 and HLA-B51, SEQ ID NO: 56 restricted to HLA-B35, SEQ ID NO: 59 restricted to HLA-B44, SEQ ID NO: 60 restricted to HLA-B44, SEQ ID NO: 61, restricted to HLA-A1 and HLA-A29, SEQ ID NO: 62 restricted to HLA-B44, SEQ ID NO: 63 restricted to HLA-A2, SEQ ID NO: 64 restricted to HLA-A2, SEQ ID NO: 67 restricted to HLA-B44, SEQ ID NO: 68 restricted to HLA-B40 and SEQ ID NO: 69 restricted to HLA-A3.
  • composition comprising said combination of CD8 T epitopes of the Ebola virus GP is capable of being presented by 13 of said predominant HLA I molecules (all the HLA I molecules selected except for HLA-B8) and of inducing a maximum frequency responders in vitro, i.e. in all individuals of a reference group who are responders to GP (corresponding to 60% of individuals in the reference group of the examples; see [Table 13])
  • said composition comprises at least one other epitope, in particular another CD8 T epitope, a CD4 T epitope or a B epitope of the NP and / or of the GP of the Ebola virus, or else an epitope of an antigen of another pathogen, in particular a hemorrhagic fever virus.
  • B epitopes of the Ebola virus NP mention may be made of EBOV peptides 173-187, 361 -375, 365-379, 381 -395, 417-431, 425-439, 485-499 and 505-515, said positions being indicated with reference to the sequence SEQ ID NO: 1.
  • B epitopes of the Ebola virus GP mention may be made of peptides 41 -55, 52- 66, 93-107, 112-126, 201-215, 217-231, 221 -235, 225-239, 236-250, 301-305, 309-323, 317-331, 321 -335, 325-339, 329-343, 333-347, 337-351, 341- 355, 345- 359, 381 -395, 385-399, 389-403, 393-407, 397-411 and 469-483 of EBOV, said positions being indicated with reference to the sequence SEQ ID NO: 49.
  • said composition comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of the GP.
  • Said composition advantageously comprises at least one CD8 T epitope according to the invention, the sequence of which is not included in the sequence comprising at least one CD4 T epitope as defined above, that is to say that all the residues of the CD8 T epitope are not present in the sequence comprising at least one CD4 T epitope.
  • Said CD8 T epitope is advantageously selected from the group consisting of the sequences SEQ ID NO: 12-14, 28-31, 38-41 and 46-48 of the NP and the sequences SEQ ID NO: 54, 56, 63, 64, 66 and 70 of the GP.
  • said composition comprises at least one CD8 T epitope according to the invention, the sequence of which is separate (non-overlapping) from that comprising at least one CD4 T epitope; this means that the sequence of the CD8 T epitope does not have any amino acid residue in common the sequence comprising at least one CD4 T epitope as defined above.
  • Said CD8 T epitope according to the invention is advantageously selected from the group consisting of the sequences SEQ ID NO: 40, 41 , 47 and 48 of the NP and the sequences SEQ ID NO: 54, 63, 64, 66 and 70 of the GP.
  • said combination of CD8 T epitopes, and optionally the other epitopes as defined above are included in one or more:
  • the immunogenic or vaccine composition comprises at least one peptide derived from NP or GP or a mixture of peptides derived from NP and / or GP; said mixture advantageously comprising at most 25 peptides; preferably 5 to 15 peptides.
  • said peptide or mixture of peptides is capable of being presented by at least 8 (8, 9, 10, 11, 12, 13 or 14), preferably at least 12 (12, 13 or 14) different predominant HLA I molecules as defined above, preferably all of said predominant HLA I molecules.
  • each of said peptides of the composition is preferably a peptide of at most 100 amino acids derived from the NP of sequence SEQ ID NO: 1 comprising at least two to four sequences comprising at least one CD8 T epitope of the NP chosen from the sequences SEQ ID NO: 2 to 48 or a peptide of at most 100 amino acids derived from the GP of sequence SEQ ID NO: 49 comprising at least two to four sequences comprising at least one CD8 T epitope of the GP chosen from the sequences SEQ ID NO: 50 to 73.
  • said peptide comprises 3 to 10 (3, 4, 5, 6, 7, 8, 9, 10) of said sequences comprising at least one CD8 T epitope according to the invention.
  • said composition comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of GP, said T CD4 and T CD8 epitope (s) being in the same peptides or in separate peptides and said composition advantageously comprising at least one CD8 T epitope whose sequence is not included in the sequence comprising at least one CD4 T epitope of NP or GP as defined above, preferably a CD8 T epitope, the sequence of which does not overlap with the sequence comprising at least one CD4 T epitope of NP or GP as defined above.
  • each of said peptides of the composition preferably consists of a sequence of more than 20 amino acids with at most 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 consecutive amino acids) of the sequence SEQ ID NO: 1 or SEQ ID NO: 49 , preferably from 25 to 70 consecutive amino acids of the sequence SEQ ID NO: 1 or SEQ ID NO: 49 or a sequence of more than 20 amino acids with at most 100 amino acids (21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids), preferably from 25 to 70 acids amines having at least 70% identity, preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99%% identity with the sequence SEQ ID NO: 1 or SEQ ID NO : 49.
  • a preferred composition according to the invention comprises at least one peptide derived from said NP selected from the group consisting of:
  • sequences of at most 100 amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids
  • sequence SEQ ID NO: 1 preferably from 25 to 70 consecutive amino acids of the sequence SEQ ID NO: 1 comprising at least one of said sequences in a)
  • said composition comprises peptides of sequence SEQ ID NO: 149, 150, 151 and 152 or peptides derived from said sequences as defined in b) or c).
  • the peptides of sequence SEQ ID NO: 149, 150, 151 and 152 induce frequencies of in vitro CD8 + T responders specific for the NP of the Ebola virus, of 36%, 16%, 44% and 58%, respectively.
  • the peptides of sequence SEQ ID NO: 149, 150 and 151 also induce frequencies of in vitro CD4 + T responders specific for the NP of the Ebola virus, of respectively 56.3%, 37.5% and 68.75% ([ Table 12]).
  • said composition also comprises the peptides of sequence SEQ ID NO: 80 and 135; the peptide of sequence SEQ ID NO: 80 induces in vitro responder frequencies in specific CD8 + T and in CD4 + T of the NP of the Ebola virus of 36% and 75% respectively.
  • the peptide of sequence SEQ ID NO: 135 induces a frequency of in vitro T-responders CD4 + specific for the NP of the Ebola virus of 62.5% ([Table 12]).
  • composition comprising the NP peptides of sequence SEQ ID NO: 80, 135, 149, 150, 151 and 152; this composition is capable of inducing a frequency of specific in vitro CD4 + T responders of 100% and a maximum frequency of specific in vitro CD8 + T responders (92% of the individuals of the reference group).
  • a composition makes it possible to obtain a maximum potential vaccination coverage of the world population and in particular of the Caucasian and African populations, as regards cellular immunity against the Ebola virus.
  • composition according to the invention comprises at least one peptide derived from said GP selected from the group consisting of:
  • sequences of not more than 100 amino acids 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids
  • sequence SEQ ID NO: 1 preferably from 25 to 70 amino acids having at least 70%, identity, preferably at least 75%, 80%, 85% 90%, 95%, 98% or 99%% identity with said sequences in a).
  • said composition comprises peptides of sequence SEQ ID NO: 153, 154, 155 and 156 or peptides derived from said sequences as defined in b) or c).
  • the peptides of sequence SEQ ID NO: 153, 154, 155 and 156 induce in vitro CD8 + T-responder frequencies specific for the GP of the Ebola virus, of 12%, 24%, 24% and 20%, respectively.
  • the peptides of sequence SEQ ID NO: 153, 154, 155 and 156 also induce frequencies of in vitro T-responders CD4 + specific for the GP of the Ebola virus, respectively 56.3%, 50%, 62.5% and 62 , 5% ([Table 13]).
  • said composition also comprises the peptides of sequence SEQ ID NO: 107 and 148; these peptides induce a frequency of in vitro T-responders CD4 + specific for the GP of the Ebola virus of 50% and 62.5% respectively for the peptides of sequence SEQ ID NO: 107 and 148.
  • this preferred composition As an illustrative example of this preferred composition according to the invention, mention may be made of the composition comprising the GP peptides of sequence SEQ ID NO: 107, 148, 153, 154, 155 and 156; this composition is capable of inducing a frequency of specific in vitro CD4 + T responders of 100% and a frequency of specific in vitro CD8 + T responders of 56%, close to the maximum frequency (60% of individuals in the reference group).
  • Such a composition makes it possible to obtain a maximum potential vaccination coverage of the world population, and in particular of the Caucasian and African populations, with regard to cellular immunity against the Ebola virus. .
  • the immunogenic or vaccine composition comprises a chimeric multi-epitope polypeptide comprising at least one combination of epitopes as defined above.
  • a chimeric polypeptide is defined here as a succession of amino acids which is not present in nature.
  • a chimeric multi-epitope polypeptide according to the invention comprises at least two to four CD8 T epitopes of the NP and / or of the GP of the Ebola virus as defined above, the sequences of said epitopes being in said polypeptide, adjacent, linked by a linking element or separated by a sequence comprising another epitope as defined above.
  • the other epitope is in particular another CD8 T epitope, a CD4 T epitope or a B epitope of the NP and / or of the GP of the Ebola virus, or else an epitope of an antigen of another pathogen, in particular a virus. hemorrhagic fever.
  • the multiepitope polypeptide can also comprise several copies of the same peptide / epitope sequence.
  • the Multiepitope polypeptide comprises less than 100 consecutive amino acids of the NP of sequence SEQ ID NO: 1 and / or of the GP of sequence SEQ ID NO: 49, preferably less than 50, more preferably less than 30.
  • the multiepitope polypeptide has 20 to 200 amino acids in length, preferably 30 to 100 amino acids, more preferably about 50 amino acids.
  • said peptide or polypeptide as defined above is a peptide or a modified polypeptide comprising a modification at the level of amino acid residue (s), of the peptide bond or its ends and said modified peptide or polypeptide retaining its immunogenic CD8 T epitope properties of Ebola virus NP or GP as defined above, that is to say that it is capable of induce a response in specific human CD8 T lymphocytes in human individuals expressing at least one of the predominant HLA I molecules as defined above.
  • This or these modification (s), preferably one or more chemical modification (s), which are introduced into the peptides by conventional methods known to those skilled in the art, include, without limitation, one of the following: less of the following chemical modifications: acetylation of the N-terminal amino acid residue and / or amidation of the C-terminal amino acid residue (Maillère et al., Molecular Immunology, 1195, 32, 1377-1385) , the substitution of an amino acid with a non-proteinogenic amino acid (D-amino acid or amino acid analog); the addition of a chemical group (lipid, oligo or polysaccharide) at the level of a reactive function, in particular of the side chain R; modification of the peptide bond (-CO-NH-), in particular by a bond of the retro or retro-inverso type (-NH-CO-) or a bond other than the peptide bond; cyclization; fusing the sequence of said peptide with that of a peptide (epitope of interest for vaccination
  • modified peptide or polypeptide it comprises one or more N6-acetyl-lysine (s), phosphoserine (s) and / or phosphothreonine (s).
  • said modified peptide or polypeptide comprises the acetylation of its N-terminal amino acid residue and / or the amidation of its C-terminal amino acid residue.
  • said modified peptide or polypeptide is a lipopeptide or a lipopolypeptide.
  • Said lipopeptide or a lipopolypeptide can be obtained, in particular by adding a lipid to an ⁇ -amino function or to a reactive function of the side chain of an amino acid of said peptide or polypeptide; it may comprise one or more chains derived from C4-20 fatty acids, optionally branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-amino hexadecanoic acid, pimelautide, trimetalside) or a derivative of a steroid.
  • the preferred lipid part is in particular represented by an N ⁇ -acetyl-lysine N E (palmitoyl) group, also called Ac-K (Pam).
  • the combination of epitopes is included in a fusion protein (chimeric protein) comprising said peptide or peptides fused with a heterologous protein (other than NP or GP) or a heterologous polypeptide fragment (fragment of a protein other than NP or GP, the sequence of which is not directly adjacent to the sequence of said peptide in the sequence of said NP or GP).
  • Said peptide (s) may be fused with the NH2 or COOH end of said protein or said heterologous polypeptide fragment or inserted into the sequence of said protein or said fragment.
  • said chimeric protein consists of one or more peptides or polypeptides as defined above, fused with one of the chains of an HLA molecule, preferably the alpha chain of an HLA molecule. I, or else with a fragment thereof corresponding to a soluble HLA molecule, in particular a fragment corresponding to the extracellular domain preceded by the homologous signal peptide or by a signal peptide heterologous.
  • Said peptide is advantageously inserted between the signal peptide and the NH 2 end of the extracellular domain of the ⁇ chain, as described for the HLA-A2 molecule (Oved K et al. Cancer Immunol Immunother (2005) 54: 867- 879).
  • a subject of the present invention is also an immunogenic or vaccine composition
  • Said polynucleotide is DNA, RNA or a mixture of DNA and RNA, recombinant or synthetic.
  • the DNA sequence can advantageously be modified so that the use of codons is optimal in the host in which it is expressed.
  • polynucleotides can be inserted into an expression vector, under the transcriptional control of an appropriate promoter, to allow the expression of said peptide (s) or polypeptide (s) in accordance with the invention in a cell from the host.
  • vectors are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or else integration into the chromosomal material of the host), as well as the nature of the host cell.
  • naked nucleic acids DNA or RNA
  • viral or bacterial vectors can be used.
  • the viral vectors are in particular adenoviruses, retroviruses, lentiviruses and AAVs in which the sequence of interest has been inserted beforehand; it is also possible to combine said sequence (isolated or inserted into a plasmid vector) with a substance allowing it to cross the membrane of host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or else the 'introducing into said host cell using physical methods such as electroporation or microinjection. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes.
  • said vector is an expression vector comprising all the elements necessary for the expression of the peptide, polypeptide, fusion protein as defined above.
  • said vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate transcriptional and possibly translation regulatory sequences (promoter, activator, intron, initiation codon ( ATG), stop codon, polyadenylation signal, splice site).
  • said composition comprises at least one polynucleotide encoding said peptide (s), polypeptide (s), and / or fusion protein (s) as defined above , inserted into naked nucleic acid, preferably naked DNA.
  • compositions in accordance with the invention can also comprise a modified antigen presenting cell, comprising at least one peptide, polypeptide, chimeric protein, polynucleotide and / or a vector as defined above, the cell being able to be modified in a stable or transient manner.
  • the cell is in particular a natural antigen presenting cell such as a dendritic cell or an artificial antigen presenting cell, such as exosomes derived from dendritic cells or vesicles derived from cells expressing NP or GP of the Ebola virus. or immunogenic fragments of said proteins.
  • the composition according to the invention can comprise a dendritic cell loaded with at least one peptide, polypeptide or chimeric protein according to the invention, or transformed with at least one polynucleotide or a vector according to the invention.
  • the immunogenic or vaccine composition according to the invention advantageously comprises a pharmaceutically acceptable vehicle, a carrier substance and / or an adjuvant.
  • compositions are those conventionally used.
  • the adjuvants are advantageously chosen from: oily emulsions, mineral substances, bacterial extracts, oligonucleotides containing CpGs, saponin, alumina hydroxide, monophosphoryl - lipid A and squalene.
  • the carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, solid microspheres of saccharide nature (poly (lactide-co-glycolide )) or gold, and nano-particles.
  • composition in accordance with the invention may further comprise adjuvants usually used in vaccines, and for example making it possible to promote the administration of the active principle, to stabilize it, to increase its immunogenicity.
  • the composition further comprises an antigen of the Ebola virus, in particular GP, in the form of a recombinant protein, of a polynucleotide or of a recombinant vector encoding said antigen.
  • said Ebola antigen optionally being combined with an adjuvant.
  • the composition according to the invention advantageously comprises a combination of T CD8 and T CD4 epitopes of the NP of the Ebola virus and a recombinant GP protein of the Ebola virus, a polynucleotide, and / or a vector encoding said antigen, optionally combined with an adjuvant. .
  • the immunogenic or vaccine composition comprises an effective dose of one or more peptide (s), polypeptide (s), protein (s), polynucleotide (s), and / or vector (s), making it possible to obtain a preventive effect on infection with an Ebola virus.
  • This dose is determined and adjusted according to factors such as the age, sex and weight of the subject.
  • the composition is generally administered according to the usual vaccination protocols, at doses and for a period sufficient to induce a cellular response in CD8 + T lymphocytes, and optionally CD4 + T lymphocytes directed against the NP and / or GP protein of the Ebola virus.
  • the administration can be oral, parenteral or local, preferably by injection, in particular subcutaneous or intramuscular.
  • the composition is in a galenic form suitable for a chosen administration.
  • the composition according to the present invention is advantageously used as a vaccine for the prevention of infections by the Ebola virus, in particular Ebola Zaire, Sudan, Reston, Bundibuygo and Tai Forest; preferably Ebola Zaire.
  • a subject of the present invention is also a method of vaccination against the Ebola virus, characterized in that it comprises the administration of a vaccine composition as defined above, to an individual, by any suitable means such as as defined above.
  • composition according to the invention induces a cellular response in CD8 T lymphocytes, and optionally in CD4 T lymphocytes, directed against the NP and / or GP protein of the Ebola virus.
  • said immunogenic or vaccine composition is used in combination with another vaccine composition against the Ebola virus, in particular a composition comprising a recombinant GP protein or a polynucleotide or vector encoding said protein.
  • Said vaccine compositions are used simultaneously, separately or sequentially.
  • a subject of the present invention is also the use of the immunogenic or vaccine composition according to the present description for the preparation of a medicament (vaccine) intended for the prevention of infections by the Ebola virus.
  • a subject of the present invention is also the in vitro use of the composition as defined above as a reagent for the diagnosis of an infection by the Ebola virus.
  • a subject of the present invention is also the in vitro use of the composition as defined above as a reagent for the immunomonitoring of the cellular response against the Ebola virus in an individual who has been exposed to the Ebola virus or who has been vaccinated against Ebola virus.
  • the present invention also relates to an in vitro method for immunomonitoring the cellular response against the Ebola virus in an individual who has been exposed to the Ebola virus or who has been vaccinated against the Ebola virus, characterized in that it includes: - bringing a biological sample of said individual comprising CD8 + T lymphocytes or a mixture of CD8 + T and CD4 + T lymphocytes into contact with a composition according to the invention as defined above, and
  • CD8 + T lymphocytes and optionally of CD4 + T lymphocytes, specific for the Ebola virus, by any appropriate means.
  • the individual is preferably a human individual.
  • the biological sample is in particular whole blood, isolated peripheral blood mononuclear cells (PBMC), lymphocytes, CD8 + T lymphocytes or a mixture of isolated CD8 + and CD4 + T lymphocytes, in particular from PBMC.
  • PBMC peripheral blood mononuclear cells
  • a subject of the present invention is also a diagnostic or immunomonitoring reagent comprising the composition as defined above.
  • said reagent comprises at least one peptide, polypeptide or chimeric protein as defined above, for example labeled and / or complexed with an HLA molecule, in particular complexed with labeled HLA molecules, for example biotinylated, in the form of HLA / peptide multimeric complexes such as tetramers of HLA / peptide complexes, labeled.
  • Said labeled HLA molecules are in particular labeled HLA I molecules, a mixture of labeled HLA I molecules or a mixture of labeled HLA I and HLA II molecules.
  • said reagent is included in a box (kit).
  • the present invention thus further relates to a method for analyzing CD8 + T lymphocytes or a mixture of CD8 + and CD4 + T lymphocytes, specific for the Ebola virus, characterized in that it comprises at least the following steps:
  • a cell sample comprising CD8 + T lymphocytes or a mixture of CD8 + and CD4 + T lymphocytes with multimeric HLA / peptide complexes, labeled, in particular with a fluorochrome, said complexes being formed by the binding of soluble HLA molecules with at least one peptide, polypeptide or chimeric protein derived from the NP or GP of the Zaire Ebola virus according to the invention, and
  • the analysis of the cells comprises sorting said cells.
  • a subject of the present invention is also a peptide consisting of an immunogenic CD8 T epitope of the NP or GP of the Ebola virus of sequence SEQ ID NO: 3, 4, 6, 9-13, 15-16, 18, 20, 22-28, 30-34, 37, 39-42, 44, 46, 47, 50 to 73 and 150 to 156 as defined above.
  • the peptides and their derivatives as defined above are prepared by standard techniques known to those skilled in the art.
  • the peptides and their derivatives can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85: 2149-) or in liquid phase, and purified by reverse phase high performance liquid chromatography.
  • the lipopeptides can in particular be prepared according to the process described in International Applications WO 99/40113 or WO 99/51630.
  • the peptides and their derivatives as defined above can also be produced from the corresponding cDNAs, obtained by any means known to those skilled in the art; the cDNA is cloned into a eukaryotic or prokaryotic expression vector and the protein or the fragment produced in the cells modified by the recombinant vector are purified by any suitable means, in particular by affinity chromatography.
  • the polynucleotides according to the invention are obtained by conventional methods, known in themselves. For example, they can be obtained by amplification of a nucleic acid sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or else by total or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.
  • FIG. 1 shows the allele frequencies (%) of the HLA I molecules selected in different Caucasian (France) and African (Cameroon, Sudan, South Africa) populations and in the reference group tested (panel).
  • the sequences of the NP (UniProt - AAD14590.1; SEQ ID NO: 1) and of the GP (UniProt - AKG65268.1; SEQ ID NO: 49) of the Ebola virus come from the American Center for Biotechnological Information ( NCBI). The Zaire strain from 1976 was used (ZEBOV). Peptides were selected using the MHC-I binding prediction tool, NetMHC v4.0. For the HLA-A * 02 allele, peptides with a rank less than or equal to 1% were selected. For the other alleles, only the ten best peptides (NP and GP combined) were selected. The 9-mer and 10-mer peptides were synthesized by Pepscan (Netherlands), with a minimum purity of 80%.
  • the blood samples come from the French Blood Establishment (Rungis Center).
  • PBMCs were isolated by ficoll gradient.
  • Immature dendritic cells (DC) were obtained from PBMCs by differentiation of adherent cells after 5 days of culture in AIM-V medium containing 1000 U / ml of IL-4 and 1000 U / ml of GM-CSF (R&D Systems).
  • Mature DCs were obtained from immature DCs after being cultured for two days in the presence of LPS.
  • the CD8 + T cells were purified from PBMCs using magnetic microbeads targeting all non-CD3 + / CD8 + populations (Miltenyi Biotec). The genotyping of the donors was carried out by NGS by the company DKMS Life Science (Dreseden, Germany).
  • the CD8 + T lymphocytes (1 to 3 ⁇ 10 5 ) were cultured in a 96-well round-bottom plate with mature autologous DCs previously loaded with pools of peptides (10 mg / ml).
  • the culture is carried out in IMDM medium supplemented with Serum AB (complete IMDM medium) containing 30 ng / ml of IL-21 (Bio-techne). 20 lymphocyte lines were seeded for each peptide pool.
  • the culture is stimulated after one week by adding 10,000 to 30,000 DC loaded with the peptide pools, 5 ng / ml IL-15 and 5 ng / ml IL-7 (Bio-techne). Between 5 and 7 days after restimulation, a specificity test is performed by ELISpot. Each culture well constitutes a line of CD8 + T lymphocytes.
  • the anti-human IFN-g 1 -D1 K antibody is adsorbed at 2.5 mg / ml in 1X PBS (Mabtech) on 96-well Multiscreen HA plates (Millipore) by an overnight incubation at 4 ° vs. The plates are then saturated by a 2 hour incubation at 37 ° C with complete IMDM medium, CD8 + T lymphocytes are incubated for 16 hours at 37 ° C in these plates after having been washed in AIM-V medium / IL-7 (0.5ng / ml IL-7). PBMCs (50,000 cells / well) incubated in the presence of 10 mg / ml of peptides are used as presenting cells.
  • a CD8 + T lymphocyte line is considered to be specific for the antigen if the number of spots in the wells containing the antigen is at least twice as high as the well not containing the antigen, the difference between the 2 types of wells being at least greater than 25.
  • HLA-A and HLA-B alleles present in at least 5% of individuals in different populations have been sought.
  • the HLA-C alleles were not retained because of the low expression of the latter compared to the HLA-A and HLA-B alleles (Neefjes and Ploegh, Eur. J. Immunol., 1988, 18, 801-810 ).
  • NP appears to be more immunogenic than GP, the number of candidate peptides being twice as high, which is consistent with the data published by Mc Elroy et al. (McElroy et al., 2015, supra).
  • CD8 + T lymphocyte lines were obtained by coculture of CD8 + T lymphocytes with autologous dendritic cells previously loaded with pools of peptides. After amplification of the T lymphocytes, each line was tested by ELISpot for its ability to recognize pools of peptides and then in a second test, the individual peptides contained in the pool recognized by the lines.
  • [0102] and [Table 3] show the individual responses in specific CD8 + T lymphocytes against the different peptides of Ebola NP and GP, respectively. Peptides (CD8 T epitopes) recognized by CD8 + T lymphocytes specific for Ebola NP and GP are indicated. The HLA (Restriction) typing for which the donor has been tested, the number of donors tested, the number of positive donors and the percentage of responding donor per allele are also indicated. [0103] [Table 2]
  • the CD8 + T lymphocyte amplification tests are carried out using a panel of healthy donors having various HLA class I molecules. Since specific T cells are present in the blood at a low frequency, T lymphocytes are amplified by cycles of antigen stimulation. The specificity of the cells for the peptides of interest is evaluated by ELISpot. These tests make it possible to evaluate the response of the lymphocytes to the peptides and thus to select the combination of peptides having a high responder frequency.
  • the sequences of the NP (UniProt AAD14590.1; SEQ ID NO: 1) and of the GP (UniProt AKG65268.1; SEQ ID NO: 49) of the Ebola virus come from the American Center for Biotechnological Information (NCBI) .
  • NCBI American Center for Biotechnological Information
  • Zaire strain from 1976 was used (ZEBOV).
  • Peptides were selected using the MHC-II Binding Prediction Tool from the IEDB database (http://www.iedb.org/).
  • the NetMHCIIpan and Sturniolo algorithms were used to predict the binding capacity of a given peptide to selected HLA class II molecules.
  • 20-mer peptides containing the nearby cores were then defined, so that the cores were not in position 1 or in position 20 of the peptide.
  • the 20-mer peptides were synthesized by Pepscan (Netherlands), with a minimum purity of 80%.
  • the blood samples come from the French Blood Establishment (Rungis Center).
  • Peripheral blood mononuclear cells PBMC
  • Immature dendritic cells DC
  • Mature DCs were obtained from immature DCs after being cultured for two days in the presence of LPS.
  • CD4 + T lymphocytes were purified from PBMCs using magnetic microbeads coupled to anti-CD4 antibodies (Miltenyi Biotec). The genotyping of the donors was carried out by NGS by the company DKMS Life Science (Dresden, Germany). 3. Obtaining peptide-specific CD4 + T lymphocyte lines
  • the CD4 + T lymphocytes (1 to 3 ⁇ 10 5 ) were cultured in a 96-well round-bottom plate with mature autologous DCs previously loaded with pools of peptides (10 mg / ml).
  • the culture is carried out in IMDM medium supplemented with Serum AB (complete IMDM medium) containing 1000U / ml of IL-6 (R&D Systems) and 10ng / ml of IL-12 (R&D Systems). 25 lymphocyte lines were seeded for each peptide pool.
  • the culture is stimulated after one week by adding 10,000 to 30,000 DC loaded with the peptide pools, 20U / ml of IL-2 (R&D Systems) and 10ng / ml of IL-7 (R&D Systems) ). Another stimulation is made after 14 and 21 days of culture. Between 5 and 7 days after the last stimulation, a specificity test is performed by ELISpot. Each culture well constitutes a line of CD4 + T lymphocytes.
  • Anti-human IFN-g antibodies (clone 1 -D1 K, Mabtech, Sweden) were adsorbed at 2.5 mg / ml in 1X PBS (Mabtech) on 96-well Multiscreen HA plates (Millipore). After overnight incubation at 4 ° C, the plates were saturated by incubating them for 2 hours at 37 ° C with complete IMDM medium. CD4 + T lymphocytes were incubated for 16 hours at 37 ° C in these plates after being washed in AIM-V medium containing 0.5ng / ml IL-7, in the presence of presenting cells (50,000 PBMC / well ) incubated in the presence of 10 mg / ml of peptides.
  • a CD4 + T cell line is considered to be antigen specific if the number of spots in the wells containing the antigen is at least twice as high as the well not containing the antigen, the difference between the two types of well being at least greater than 25.
  • the peptides were selected using the MHC-II binding prediction tool from the IEDB database (http://www.iedb.org/). Only the HLA-DR molecules most abundantly expressed on the surface of Antigen Presenting Cells (APC) were studied. The DRB1 alleles among the most frequent in different populations [Table 7] were selected.
  • HLA-DRB3 * 01: 01, HLA-DRB3 * 02: 02, HLA-DRB4 * 01: 01 and HLA-DRB5 * 01: 01 alleles were added.
  • 15 HLA-DR alleles, representative of the Caucasian and African populations, listed below were selected: HLA-DRB1 * 01: 01, HLA-DRB1 * 03: 01, HLA-DRB1 * 04: 01, HLA-DRB1 * 04:05, HLA- DRB1 * 07: 01, HLA-DRB1 * 08: 02, HLA-DRB1 * 09: 01, HLA-DRB1 * 11: 01, HLA- DRB1 * 12: 01, HLA-DRB1 * 13 : 01, HLA- DRB1 * 15: 01, HLA-DRB3 * 01: 01, HLA- DRB3 * 02: 02, HLA-DRB4 * 01: 01 and HLA-DRB5 * 01: 01.
  • the NetMHCIIpan and Sturniolo algorithms made it possible to predict the binding capacity of a given peptide to selected HLA class II molecules.
  • a rank is determined, its predicted affinity being compared to that of 200,000 natural peptides.
  • the 9-mer (core) peptides with a rank of less than 10% for at least 4 HLA-DR alleles, or the peptides with a rank of less than 5% for at least 1 allele were selected.
  • 20-mer peptides containing the close hearts were then defined, so that the hearts were not in position 1 or in position 20 of the peptide.
  • 23 20-mer peptides for NP and 33 20-mer peptides for GP were selected to be tested in vitro. [0117] [Table 7]
  • CD4 + T lymphocyte lines were obtained by co-culture of CD4 + T lymphocytes with autologous dendritic cells previously loaded with pools of peptides. After amplification of the T lymphocytes, each line was tested by ELISpot for its ability to recognize pools of peptides and then in a second test, the individual peptides contained in the pool recognized by the lines. In order to ensure the capacity of each donor to respond, CD4 + lymphocyte lines specific for a pool of peptides specific for Cytomegalovirus, Epstein-barr and influenza were also produced.
  • [0119] [Table 8] and [Table 9] show the individual responses in specific CD4 + T lymphocytes against the different peptides of Ebola NP and GP respectively.
  • the CD4 T lymphocyte lines were obtained by in vitro stimulation by dendritic cells previously loaded with the NP or GP peptides. The specificity for these peptides was evaluated by Elispot IFN ⁇ .
  • [Table 8] and [Table 9] indicate the rate of responders (in%) and the intensity of response (percentage of positive lines) for each of the peptides.
  • Table 91 Individual response of CD4 + T lymphocytes against GP peptides [0122] It should be noted that only one peptide (GP18-37) does not induce a response in any of the donors. For each peptide, the intensity of response is variable depending on the donors, from 1 to 14 positive lines. Furthermore, the number of total positive lines per donor is also extremely variable, from 1 to 83 and from 1 to 105 for PN and GP respectively.
  • the results obtained are very heterogeneous depending on the peptides studied.
  • the data can therefore be analyzed according to two parameters: the rate (number of responder donors) and the intensity (number of total positive lines) of response for each peptide for peptides derived from NP and GP respectively.
  • Ten peptides for NP and 8 for GP induce responses in 50% or more of donors.
  • the peptides responding in the largest number of donors are also those for which the number of positive lines is the highest.
  • NP is more immunogenic than GP, 54 epitopes having been obtained for the first, and 28 for the second.
  • Ebola CD8 T epitopes are advantageously combined with the Ebola CD4 T epitopes (example 2) so as to obtain a combination of peptides allowing maximum coverage of the donors tested, both in T CD4 response and in CD8 T response against Ebola virus.
  • the combination of the T CD4 and T CD8 epitopes of Ebola thus makes it possible to increase the effectiveness of a vaccine against the Ebola virus.

Abstract

The invention concerns an immunogenic or vaccine composition against the Ebola virus, comprising a combination of CD8 T epitopes of the Ebola virus which is able to be presented by at least four different predominant HLA I molecules and to induce a specific human CD8+ T lymphocyte response in individuals expressing at least one of said HLA I modules. The invention also concerns the use of said composition as a preventative vaccine against the Ebola virus and as a reagent for immunomonitoring of the cellular response against the Ebola virus.

Description

MELANGES D’EPITOPES T CD8 IMMUNOGÈNES DU MIXTURES OF T CD8 IMMUNOGENS EPITOPES
VIRUS EBOLA EBOLA VIRUS
Domaine technique Technical area
[0001] L’invention concerne une composition immunogène ou vaccinale contre le virus Ebola, comprenant une combinaison d’épitopes T CD8 du virus Ebola qui est apte à être présentée par au moins quatre molécules HLA I prépondérantes différentes et à induire une réponse en lymphocytes T CD8 humains spécifiques chez des individus exprimant au moins l’une desdites molécules HLA I. L’invention concerne également l’utilisation de ladite composition comme vaccin préventif contre le virus Ebola et comme réactif pour l’immunomonitorage de la réponse cellulaire contre le virus Ebola. The invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising a combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules and of inducing a lymphocyte response Human CD8 T specific in individuals expressing at least one of said HLA I molecules. The invention also relates to the use of said composition as a preventive vaccine against Ebola virus and as a reagent for immunomonitoring the cellular response against the virus. Ebola.
Technique antérieure Prior art
[0002] Le virus Ebola est un virus de la famille des Filoviridae hautement virulent, responsable d’épidémies de fièvres hémorragiques mortelles, chez l’Homme et le primate non-humain. Ce virus comprend une seule protéine d’enveloppe, la Glycoprotéine (GP), ainsi que des protéines internes dont la Nucléoprotéine (NP) qui est la protéine virale la plus exprimée avec environ 3200 molécules par virion et potentiellement la plus immunogène. Cinq espèces du virus Ebola ont été identifiées: Zaïre, Soudan, Reston, Forêt de Taï et Bundibugyo. Le virus Ebola Zaïre (EBOV) est responsable de la plupart des flambées épidémiques depuis la découverte du virus Ebola en 1976, avec un taux de létalité moyen d’environ 50 % (25 % à 90 % au cours des flambées récentes). [0002] The Ebola virus is a highly virulent virus of the Filoviridae family responsible for epidemics of fatal hemorrhagic fevers in humans and non-human primates. This virus comprises a single envelope protein, Glycoprotein (GP), as well as internal proteins including Nucleoprotein (NP) which is the most expressed viral protein with around 3200 molecules per virion and potentially the most immunogenic. Five species of the Ebola virus have been identified: Zaire, Sudan, Reston, Forêt de Taï and Bundibugyo. Zaire Ebola virus (EBOV) has been responsible for most epidemic outbreaks since the discovery of Ebola virus in 1976, with an average case fatality rate of around 50% (25% to 90% in recent outbreaks).
[0003] Aucun vaccin préventif n’a été approuvé pour le traitement des infections par Ebola mais plusieurs vaccins basés sur la glycoprotéine (GP) sont en développement. Il s’agit de vecteurs viraux réplicatifs tels que le virus de la stomatite vésiculaire (rVSV-Ebov), le cytomégalovirus (rCMV), ou le virus parainfluenza humain de type 3 (HPIV3), ainsi que d’adénovirus non-réplicatifs tels que Chad3-EBOV (Chimp adenovirus), EBOLA rAd-5 et Ad26.ZEBOV. On peut citer également, des plasmides (INO-4212 ou VRC-EBODNA023-00-VP) et des nanoparticules contenant la glycoprotéine (GP) recombinante combinées à un adjuvant à base de saponine (Matrix-M™). Les essais précliniques, chez le rongeur ou le primate non-humain se sont révélés concluants, apportant la preuve de concept d’une protection virale par vaccination dans ces modèles. Des essais chez l’homme sont également en cours. [0003] No preventive vaccine has been approved for the treatment of Ebola infections, but several vaccines based on glycoprotein (GP) are in development. These are replicative viral vectors such as vesicular stomatitis virus (rVSV-Ebov), cytomegalovirus (rCMV), or human parainfluenza virus type 3 (HPIV3), as well as non-replicating adenoviruses such as Chad3-EBOV (Chimp adenovirus), EBOLA rAd-5 and Ad26.ZEBOV. Mention may also be made of plasmids (INO-4212 or VRC-EBODNA023-00-VP) and nanoparticles containing the recombinant glycoprotein (GP) combined with a saponin-based adjuvant (Matrix-M ™). Preclinical trials, in rodents or non-human primates, have proved to be conclusive, providing proof of concept for viral protection by vaccination in these models. Human trials are also underway.
[0004] Bien que prometteurs, ces vaccins présentent plusieurs limites. Initialement développés pour la lutte contre le bioterrorisme, ils ne sont pas adaptés à une vaccination à grande échelle en Afrique Sub-Saharienne. Une production massive est compliquée, et une dissémination dans les régions endémiques nécessite un stockage à -80°C pour assurer leur conservation. Par ailleurs, l’existence d’une immunité dirigée contre les vecteurs, comme les adénovirus, peut rendre le vaccin inefficace. En outre, ces vaccins ciblent uniquement la GP qui est la seule protéine du virus apte à être produite de manière recombinante pour la vaccination. [0004] Although promising, these vaccines have several limitations. Initially developed for the fight against bioterrorism, they are not suitable for large-scale vaccination in Sub-Saharan Africa. Massive production is complicated, and dissemination in endemic regions requires storage at -80 ° C to ensure their preservation. In addition, the existence of immunity against vectors, such as adenoviruses, can render the vaccine ineffective. In addition, these vaccines target only GP which is the only virus protein capable of being produced recombinantly for vaccination.
[0005] Peu d’études sont consacrées à l’immunité contre le virus Ebola et les corrélats de protection ne sont pas connus. Seule protéine de surface du virus, la GP est une cible-clé pour le développement d’une réponse humorale contre le virus. La présence d’anticorps neutralisants dirigés contre la GP confère d’ailleurs une protection et des titres élevés d’anticorps dirigés contre la GP du virus sont toujours détectables chez des patients ayant survécu à l’infection virale. La présence de lymphocytes T CD4+ (ou lymphocytes T CD4) et de lymphocytes T CD8+ (ou lymphocytes T CD8) dirigés contre les protéines virales, en particulier la NP et la GP a également été montrée chez des patients ayant survécu à l’infection virale (McElroy ét al., Proc. Natl Soi., 2015, 112, 4719-4724). Compte tenu du rôle majeur des lymphocytes T CD4+ dans l’initiation des réponses humorales et cytotoxiques, la réponse des lymphocytes T CD4 est nécessaire à l’établissement de l’immunité anti-EBOLA mais les caractéristiques de cette réponse sont peu connues. Les lymphocytes T CD8+ jouent un rôle majeur dans la réponse immunitaire contre le virus Ebola. Il a en effet été montré une corrélation entre survie à l’infection et taux de lymphocytes T CD8+ activés (Sanchez et al., J. Virol. , 2004, 78, 10370-10377). Il a également été montré que des souris vaccinées avec le Virus de l'encéphalite équine vénézuélienne codant pour la NP d’Ebola développaient une réponse CD8+ spécifique de la protéine, protégeant ainsi les animaux d’une infection létale (Wilson et Hart, J. Virol. , 2001 , 75, 2660-2664). En revanche, peu de données sont disponibles sur le rôle d’une réponse cellulaire contre la GP. [0005] Few studies have been devoted to immunity against the Ebola virus and the correlates of protection are not known. The only surface protein in the virus, GP is a key target for the development of a humoral response against the virus. The presence of neutralizing antibodies directed against GP moreover confers protection and high titers of antibodies directed against the GP of the virus are always detectable in patients who have survived the viral infection. The presence of CD4 + T lymphocytes (or CD4 T lymphocytes) and CD8 + T lymphocytes (or CD8 T lymphocytes) directed against viral proteins, in particular PN and GP has also been shown in patients who have survived the viral infection (McElroy et al., Proc. Natl Soi., 2015, 112, 4719-4724). Given the major role of CD4 + T lymphocytes in the initiation of humoral and cytotoxic responses, the response of CD4 T lymphocytes is necessary for the establishment of anti-EBOLA immunity, but the characteristics of this response are little known. CD8 + T lymphocytes play a major role in the immune response against the Ebola virus. A correlation between survival to infection and the level of activated CD8 + T lymphocytes has in fact been shown (Sanchez et al., J. Virol., 2004, 78, 10370-10377). It has also been shown that mice vaccinated with the Venezuelan Equine Encephalitis Virus encoding Ebola PN developed a protein-specific CD8 + response, thereby protecting the animals from lethal infection (Wilson and Hart, J. Virol., 2001, 75, 2660-2664). In contrast, little data is available on the role of a cellular response against GP.
[0006] Les lymphocytes T CD8+ cytotoxiques (CTL) sont capables de reconnaître les antigènes d’un virus pathogène, sous la forme de peptides d’une longueur de 9-10 acides aminés, dénommés épitopes T CD8+ (ou épitope T CD8), présentés par les molécules du Complexe Majeur d’Histocompatibilité de classe I (HLA I (HLA-A, HLA-B et HLA-C) chez l’homme) à la surface des cellules infectées et de lyser les cellules infectées directement après reconnaissance des peptides. Compte-tenu du polymorphisme des molécules HLA I dans la population, la spécificité de liaison des molécules HLA I est très variable d’une molécule HLA de classe I à l’autre. De ce fait, la séquence des épitopes T CD8+ varient d’un individu à l’autre en fonction des molécules HLA-I codées par leurs allèles HLA I, dénommées ci-après allèles HLA I. L’ensemble des allèles HLA I d’un individu est dénommé typage HLA I. Par conséquent, pour élaborer un vaccin efficace, il est indispensable que celui-ci contiennent des épitopes T CD8+ pouvant être présentés par des molécules HLA I différentes afin d’avoir une couverture vaccinale la plus large possible. [0006] Cytotoxic CD8 + T lymphocytes (CTL) are capable of recognizing the antigens of a pathogenic virus, in the form of peptides with a length of 9-10 amino acids, called CD8 + T epitopes (or CD8 T epitope), presented by molecules of the Class I Major Histocompatibility Complex (HLA I (HLA-A, HLA-B and HLA-C) in humans) on the surface of infected cells and lyse the infected cells directly after recognition of the peptides. Due to the polymorphism of HLA I molecules in the population, the binding specificity of HLA I molecules is highly variable from one HLA class I molecule to another. Therefore, the sequence of CD8 + T epitopes vary from one individual to another depending on the HLA-I molecules encoded by their HLA I alleles, hereinafter referred to as HLA I alleles. All of the HLA I alleles of an individual is referred to as HLA I typing. Consequently, in order to develop an effective vaccine, it is essential that the latter contain CD8 + T epitopes which can be presented by different HLA I molecules in order to have the widest possible vaccination coverage.
Problème technique Technical problem
[0007] Très peu d’épitopes T CD8 du virus Ebola immunogènes chez l’Homme ont été caractérisés jusqu’à présent et ces derniers sont tous issus de la NP et restreints uniquement à HLA-A2 (NP 150-158), HLA-A23 (NP 82-90) et HLA-B35 (NP 313-321 ) (Ruibal ét al., Nature, 2016, 533, 100-104). Pour mettre au point des vaccins efficaces contre le virus Ebola permettant d’avoir une couverture vaccinale la plus large possible, il existe donc un réel besoin de disposer d’un ensemble d’épitopes T CD8 de la NP et de la GP du virus Ebola capables d’être présentés par des molécules HLA I différentes représentatives de la population à vacciner et d’induire une réponse en lymphocytes T CD8 humains spécifiques chez les individus exprimant lesdites molécules HLA I. Résumé de l’invention [0007] Very few CD8 T epitopes of the immunogenic Ebola virus in humans have been characterized so far and they are all derived from NP and restricted only to HLA-A2 (NP 150-158), HLA- A23 (NP 82-90) and HLA-B35 (NP 313-321) (Ruibal et al., Nature, 2016, 533, 100-104). To develop effective vaccines against the Ebola virus allowing for the widest possible vaccination coverage, there is therefore a real need for a set of CD8 T epitopes of the NP and GP of the Ebola virus. capable of being presented by different HLA I molecules representative of the population to be vaccinated and of inducing a response in specific human CD8 T lymphocytes in individuals expressing said HLA I molecules. Summary of the invention
[0008] Les inventeurs ont identifié de nombreux épitopes T CD8 de la NP et de la GP du virus Ebola Zaïre capables de stimuler des lymphocytes T CD8 spécifiques chez un panel de donneurs sains représentatifs des populations caucasiennes et africaines ([Tableau 2], [Tableau 3] et [Tableau 4] ; Figure 1 ). Contrairement aux épitopes T CD8 de l’art antérieur immunogènes chez l’homme qui sont restreints à seulement 3 molécules HLA I prépondérantes (HLA-A2, HLA-A23, HLA-B35), ces épitopes T CD8 sont immunogènes chez des individus humains portant des molécules HLA I variées incluant la totalité des molécules HLA-A et HLA-B les plus fréquentes dans les populations caucasiennes et africaines (HLA-A1 , -A2, - A3, -A11 , -A24, -A29, -B7, -B8, -B15, -B18, -B35, -B40, -B44 et -B51 ) à l’exception de HLA-A23 ([Tableau 1 ]).. Cette ensemble d’allèles HLA-A et HLA-B permet d’obtenir une couverture vaccinale de potentiellement 95.2% des individus de la population mondiale ; 98,7% des individus d’Europe ; entre 67,9 et 80,9% des individus d’Afrique ; entre 78,8% et 92,2% des individus d’Asie et d’Océanie ; et entre 73,4% à 95,1 % des individus d’Amérique et des Caraïbes. La NP est plus immunogène que la GP, 54 épitopes ayant été obtenus pour la première et 28 pour la seconde, en accord avec les observations précédentes chez des patients ayant été infectés par le virus (McElroy 2015, précité). De plus la séquence de la NP est plus conservée et les épitopes T CD8 issus de la NP sont donc potentiellement efficaces contre les différentes souches d’Ebola. En combinant plusieurs épitopes T CD8 de l’invention, et éventuellement des épitopes T CD4, dans un ou plusieurs peptides/polypeptides issus de la NP ou de la GP du virus Ebola, polypeptides multiépitopiques ou protéines chimériques, et/ou polynucléotides, vecteurs recombinants, on obtient ainsi un vaccin contre le virus Ebola qui possède une couverture vaccinale potentielle optimale dans les différentes populations, en particulier africaines et caucasiennes. Onze épitopes de la NP et 10 épitopes de la GP permettent une couverture vaccinale maximale dans la population de référence (100% des individus répondeurs correspondant à 92% et 60% des individus de la population, respectivement pour les peptides de laThe inventors have identified numerous CD8 T epitopes of NP and GP of the Zaire Ebola virus capable of stimulating specific CD8 T lymphocytes in a panel of healthy donors representative of Caucasian and African populations ([Table 2], [ Table 3] and [Table 4]; Figure 1). Unlike prior art CD8 T epitopes which are immunogenic in humans which are restricted to only 3 predominant HLA I molecules (HLA-A2, HLA-A23, HLA-B35), these CD8 T epitopes are immunogenic in human individuals carrying various HLA I molecules including all of the most frequent HLA-A and HLA-B molecules in Caucasian and African populations (HLA-A1, -A2, - A3, -A11, -A24, -A29, -B7, - B8, -B15, -B18, -B35, -B40, -B44 and -B51) with the exception of HLA-A23 ([Table 1]) .. This set of HLA-A and HLA-B alleles makes it possible to '' achieve vaccination coverage of potentially 95.2% of individuals in the world population; 98.7% of people in Europe; between 67.9 and 80.9% of people in Africa; between 78.8% and 92.2% of individuals from Asia and Oceania; and between 73.4% to 95.1% of people in America and the Caribbean. PN is more immunogenic than GP, 54 epitopes having been obtained for the first and 28 for the second, in agreement with previous observations in patients who have been infected with the virus (McElroy 2015, supra). In addition, the NP sequence is more conserved and the CD8 T epitopes derived from NP are therefore potentially effective against the various strains of Ebola. By combining several CD8 T epitopes of the invention, and optionally CD4 T epitopes, in one or more peptides / polypeptides derived from the NP or the GP of the Ebola virus, multepitope polypeptides or chimeric proteins, and / or polynucleotides, recombinant vectors , we thus obtain a vaccine against the Ebola virus which has an optimal potential vaccination coverage in the various populations, in particular African and Caucasian. Eleven NP epitopes and 10 GP epitopes allow maximum vaccination coverage in the reference population (100% of responder individuals corresponding to 92% and 60% of individuals in the population, respectively for the peptides of the
NP et les peptides de la GP). En combinant plusieurs épitopes T CD8 et T CD4 dans de longs fragments polypeptidiques (LSP), 6 LSP pour la NP comme pour la GP, on obtient une couverture vaccinale potentielle maximale de la population de référence, pour les réponses en lymphocytes T CD8 et T CD4 spécifiques du virus Ebola ([Tableau 12] et [Tableau 13]). NP and GP peptides). By combining several T CD8 and T CD4 epitopes in long polypeptide fragments (LSP), 6 LSPs for both NP and GP, we obtain a maximum potential vaccination coverage of the reference population, for the responses in CD8 T lymphocytes and CD4 T lymphocytes specific for the Ebola virus ([Table 12] and [Table 13]).
[0009] La présente invention a pour objet une composition immunogène ou vaccinale contre le virus Ebola, comprenant au moins une combinaison d’épitopes T CD8 du virus Ebola qui est apte à être présentée par au moins quatre molécules HLA I prépondérantes différentes choisies parmi HLA-A1 , HLA-A2, HLA-A3, HLA- A11 , HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA- B40, HLA-B44 et HLA-B51 et à induire une réponse en lymphocytes T CD8+ humains spécifiques chez des individus exprimant au moins l’une desdites molécules HLA-I, et lesdits épitopes étant sélectionnés dans le groupe constitué par : The present invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising at least one combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules chosen from HLA -A1, HLA-A2, HLA-A3, HLA- A11, HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA- B40, HLA-B44 and HLA-B51 and inducing a specific human CD8 + T lymphocyte response in individuals expressing at least one of said HLA-I molecules, and said epitopes being selected from the group consisting of:
- les épitopes SEQ ID NO : 28 et 46 de la nucléoprotéine (NP), restreints à HLA- A1 - the epitopes SEQ ID NO: 28 and 46 of the nucleoprotein (NP), restricted to HLA-A1
- les épitopes SEQ ID NO : 2, 5, 7, 8, 11 , 13, 14, 17, 20, 23, 36, 38 et 45 de la NP et SEQ ID NO : 53, 57, 58, 63 , 64, 65, 72 et 73 de la glycoprotéine (GP), restreints à HLA-A2 - the epitopes SEQ ID NO: 2, 5, 7, 8, 11, 13, 14, 17, 20, 23, 36, 38 and 45 of NP and SEQ ID NO: 53, 57, 58, 63, 64, Glycoprotein (GP) 65, 72 and 73, restricted to HLA-A2
- les épitopes SEQ ID NO : 9 et 44 de la NP et SEQ ID NO : 69 de la GP, restreints à HLA-A3 - the epitopes SEQ ID NO: 9 and 44 of the NP and SEQ ID NO: 69 of the GP, restricted to HLA-A3
- les épitopes SEQ ID NO : 24 de la NP et SEQ ID NO : 71 de la GP, restreints à HLA-A11 - the epitopes SEQ ID NO: 24 of the NP and SEQ ID NO: 71 of the GP, restricted to HLA-A11
- les épitopes SEQ ID NO : 16 de la NP et SEQ ID NO : 50 et 51 de la GP restreints à HLA-A24 - the epitopes SEQ ID NO: 16 of the NP and SEQ ID NO: 50 and 51 of the GP restricted to HLA-A24
- les épitopes SEQ ID NO : 10 et 41 de la NP, restreints à HLA-A29 - the epitopes SEQ ID NO: 10 and 41 of NP, restricted to HLA-A29
- les épitopes SEQ ID NO : 33 de la NP, restreint à HLA-B7 - the epitopes SEQ ID NO: 33 of NP, restricted to HLA-B7
- les épitopes SEQ ID NO : 26 de la NP, restreint à HLA-B8 - the epitopes SEQ ID NO: 26 of NP, restricted to HLA-B8
- les épitopes SEQ ID NO : 12, 15, 22, 29 et 37 de la NP et SEQ ID NO : 52 de la - the epitopes SEQ ID NO: 12, 15, 22, 29 and 37 of the NP and SEQ ID NO: 52 of the
GP, restreints à HLA-B15 GP, restricted to HLA-B15
- G épitope SEQ ID NO : 25 de la NP, restreint à HLA-B18 - G epitope SEQ ID NO: 25 of NP, restricted to HLA-B18
- les épitopes SEQ ID NO : 19, 40, 43, 48 de la NP et SEQ ID NO : 56 de la GP, restreints à HLA-B35 - the epitopes SEQ ID NO: 19, 40, 43, 48 of the NP and SEQ ID NO: 56 of the GP, restricted to HLA-B35
- les épitopes SEQ ID NO : 3, 30, 39 et 47 de la NP et SEQ ID NO : 60 et 68 de la GP, restreints à HLA-B40 - the epitopes SEQ ID NO: 3, 30, 39 and 47 of the NP and SEQ ID NO: 60 and 68 of the GP, restricted to HLA-B40
- les épitopes SEQ ID NO : 4 et 31 de la NP et SEQ ID NO : 59, 62, 66 et 67 de la GP, restreints à HLA-B44 - the epitopes SEQ ID NO: 4 and 31 of the NP and SEQ ID NO: 59, 62, 66 and 67 of the GP, restricted to HLA-B44
- les épitopes SEQ ID NO : 18 et 32 de la NP et SEQ ID NO : 70 de la GP, restreints à HLA-B51 - the epitopes SEQ ID NO: 18 and 32 of the NP and SEQ ID NO: 70 of the GP, restricted to HLA-B51
- l’épitope SEQ ID NO : 61 de la GP, restreint à HLA-A1 et HLA-A29 - the epitope SEQ ID NO: 61 of the GP, restricted to HLA-A1 and HLA-A29
- G épitope SEQ ID NO : 21 de la NP, restreint à HLA-A2 et HLA-A24 - G epitope SEQ ID NO: 21 of NP, restricted to HLA-A2 and HLA-A24
- G épitope SEQ ID NO : 6 de la NP, restreint à HLA-A29 et HLA-B15 - G epitope SEQ ID NO: 6 of NP, restricted to HLA-A29 and HLA-B15
- l’épitope SEQ ID NO : 35 de la NP, restreint à HLA-B7 et HLA-B35 - the epitope SEQ ID NO: 35 of NP, restricted to HLA-B7 and HLA-B35
- l’épitope SEQ ID NO : 27 de la NP, restreint à HLA-B7 et HLA-B51 - the epitope SEQ ID NO: 27 of NP, restricted to HLA-B7 and HLA-B51
- l’épitope SEQ ID NO : 55 de la GP, restreint à HLA-B7, HLA-B18 et HLA-B51 - the epitope SEQ ID NO: 55 of the GP, restricted to HLA-B7, HLA-B18 and HLA-B51
- les épitopes SEQ ID NO : 34 et 42 de la NP, restreints à HLA-B18 et HLA-B40, et - the epitopes SEQ ID NO: 34 and 42 of NP, restricted to HLA-B18 and HLA-B40, and
- G épitope SEQ ID NO : 54 de la GP, restreint à HLA-B40 et HLA-B44. [0010] Dans des modes de réalisation particuliers, ladite composition immunogène ou vaccinale comprend une combinaison d’épitopes choisie de manière à ce qu’elle soit apte à être présentée par au moins 8 molécules HLA I prépondérantes différentes, de préférence au moins 12, 13 ou la totalité desdites molécules HLA I prépondérantes. - G epitope SEQ ID NO: 54 of GP, restricted to HLA-B40 and HLA-B44. [0010] In particular embodiments, said immunogenic or vaccine composition comprises a combination of epitopes chosen so that it is suitable for being presented by at least 8 different predominant HLA I molecules, preferably at least 12, 13 or all of said predominant HLA I molecules.
[0011] Dans des modes de réalisation particuliers, ladite composition comprend une combinaison d’épitopes sélectionnés dans le groupe constitué par : les séquences SEQ ID NO : 3 à 7, 9 à 22, 24 à 35 et 37 à 48 de la NP et les séquences SEQ ID NO : 50 à 52, 54 à 56 et 59 à 64 et 66 à 71 de la GP. De préférence, la composition immunogène ou vaccinale comprend au moins un épitope sélectionné dans le groupe constitué par : les séquences SEQ ID NO : 5, 6, 10, 15, 17, 24, 25, 34, 41 et 42 de la NP et les séquences SEQ ID NO : 55, 61 et 71 de la GP. [0011] In particular embodiments, said composition comprises a combination of epitopes selected from the group consisting of: the sequences SEQ ID NO: 3 to 7, 9 to 22, 24 to 35 and 37 to 48 of the NP and the sequences SEQ ID NO: 50 to 52, 54 to 56 and 59 to 64 and 66 to 71 of the GP. Preferably, the immunogenic or vaccine composition comprises at least one epitope selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 15, 17, 24, 25, 34, 41 and 42 of the NP and the sequences SEQ ID NO: 55, 61 and 71 of the GP.
[0012] Dans des modes de réalisation particuliers, ladite composition immunogène ou vaccinale comprend au moins un épitope T CD8 conservé dans différentes souches du virus Ebola, choisi parmi les séquences SEQ ID NO : 3 à 8, 10 à 39, 41 , 43, 47 et 48 de la NP et les séquences SEQ ID NO : 51 à 60 et 67 à 71 de la GP; de préférence choisi parmi les séquences SEQ ID NO : 5, 6, 12, 15 à 24, 27 à 38 et 41 de la NP et les séquences SEQ ID NO : 51 , 54, 55, 57 à 60, 67 et 70 de la GP ; de manière encore plus préférée parmi les séquences SEQ ID NO : SEQ ID NO : 15 à 18, 20 à 24, 27, 30 à 33, 36 et 37 de la NP et les séquences SEQ ID NO : 55, 57, 58, 60 et 67 de la GP. In particular embodiments, said immunogenic or vaccine composition comprises at least one CD8 T epitope conserved in different strains of the Ebola virus, chosen from the sequences SEQ ID NO: 3 to 8, 10 to 39, 41, 43, 47 and 48 of the NP and the sequences SEQ ID NO: 51 to 60 and 67 to 71 of the GP; preferably chosen from the sequences SEQ ID NO: 5, 6, 12, 15 to 24, 27 to 38 and 41 of the NP and the sequences SEQ ID NO: 51, 54, 55, 57 to 60, 67 and 70 of the GP; even more preferably among the sequences SEQ ID NO: SEQ ID NO: 15 to 18, 20 to 24, 27, 30 to 33, 36 and 37 of the NP and the sequences SEQ ID NO: 55, 57, 58, 60 and 67 of the GP.
[0013] Dans des modes de réalisation particuliers, ladite composition immunogène ou vaccinale comprend une combinaison d’épitopes T CD8 de la NP ou une combinaison d’épitopes T CD8 de la NP et de la GP. [0013] In particular embodiments, said immunogenic or vaccine composition comprises a combination of CD8 T epitopes from NP or a combination of CD8 T epitopes from NP and GP.
[0014] Dans des modes de réalisation préférés, ladite composition immunogène ou vaccinale comprend une combinaison d’épitopes T CD8 de la NP comprenant au moins : [0014] In preferred embodiments, said immunogenic or vaccine composition comprises a combination of CD8 T epitopes of NP comprising at least:
a) les épitopes SEQ ID NO : 5 restreint à HLA-A2 ; SEQ ID NO : 16 restreint à HLA-A24, SEQ ID NO : 26 restreint à HLA-B8, SEQ ID NO : 28 restreint à HLA-A1 et SEQ ID NO : 31 restreint à HLA-B44 ; a) the epitopes SEQ ID NO: 5 restricted to HLA-A2; SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 28 restricted to HLA-A1 and SEQ ID NO: 31 restricted to HLA-B44;
b) l’un des épitopes SEQ ID NO : 13, SEQ ID NO : 14 ou SEQ ID NO : 17 restreint à HLA-A2 , de préférence l’épitope SEQ ID NO : 17; b) one of the epitopes SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 17 restricted to HLA-A2, preferably the epitope SEQ ID NO: 17;
c) l’un des épitopes SEQ ID NO : 9 ou SEQ ID NO : 44 restreint à HLA-A3 ; de préférence l’épitope SEQ ID NO : 44 ; c) one of the epitopes SEQ ID NO: 9 or SEQ ID NO: 44 restricted to HLA-A3; preferably the epitope SEQ ID NO: 44;
d) l’épitope SEQ ID NO : 24 restreint à HLA-A11 ; d) the epitope SEQ ID NO: 24 restricted to HLA-A11;
e) l’épitope SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15 ou l’un des épitopes SEQ ID NO : 10 ou SEQ ID NO : 41 restreint à HLA-A29 ; de préférence l’épitope SEQ ID NO : 6; e) the epitope SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15 or one of the epitopes SEQ ID NO: 10 or SEQ ID NO: 41 restricted to HLA-A29; preferably the epitope SEQ ID NO: 6;
f) l’un des épitopes SEQ ID NO : 3, SEQ ID NO : 30, SEQ ID NO : 39 ou SEQ ID NO : 47 restreint à HLA-B40 ou l’un des épitopes SEQ ID NO : 34 ou SEQ ID NO : 42 restreints à HLA-B40 et HLA-B18, de préférence l’épitope SEQ ID NO : 30, SEQ ID NO : 34 ou SEQ ID NO : 42, de manière plus préférée l’épitope SEQ ID NO : 34 ou SEQ ID NO : 42 et ; f) one of the epitopes SEQ ID NO: 3, SEQ ID NO: 30, SEQ ID NO: 39 or SEQ ID NO: 47 restricted to HLA-B40 or one of the epitopes SEQ ID NO: 34 or SEQ ID NO : 42 restricted to HLA-B40 and HLA-B18, preferably the epitope SEQ ID NO: 30, SEQ ID NO: 34 or SEQ ID NO: 42, more preferably the epitope SEQ ID NO: 34 or SEQ ID NO: 42 and;
g) l’épitope SEQ ID NO : 27 restreint à HLA-B7 et HLA-B51 , l’épitope SEQ ID NO : 33 restreint à HLA-B7 ou l’épitope SEQ ID NO : 35 restreint à HLA-B7 et HLA- B35 , de préférence l’épitope SEQ ID NO : 27 ou SEQ ID NO : 35 ; de manière plus préférée l’épitope SEQ ID NO : 27 . g) the epitope SEQ ID NO: 27 restricted to HLA-B7 and HLA-B51, the epitope SEQ ID NO: 33 restricted to HLA-B7 or the epitope SEQ ID NO: 35 restricted to HLA-B7 and HLA- B35, preferably the epitope SEQ ID NO: 27 or SEQ ID NO: 35; more preferably the epitope SEQ ID NO: 27.
[0015] Dans des modes de réalisation préférés, ladite composition immunogène ou vaccinale, comprend une combinaison d’épitopes T CD8 de la GP comprenant au moins [0015] In preferred embodiments, said immunogenic or vaccine composition comprises a combination of CD8 T epitopes of GP comprising at least
- l’épitope SEQ ID NO : 61 restreint à HLA-A1 et HLA-A29, - the epitope SEQ ID NO: 61 restricted to HLA-A1 and HLA-A29,
- l’épitope SEQ ID NO : 63 ou SEQ ID NO : 64 restreint à HLA-A2 - the epitope SEQ ID NO: 63 or SEQ ID NO: 64 restricted to HLA-A2
- l’épitope SEQ ID NO : 69 restreint à HLA-A3 - the epitope SEQ ID NO: 69 restricted to HLA-A3
- l’épitope SEQ ID NO : 71 restreint à HLA-A11 - the epitope SEQ ID NO: 71 restricted to HLA-A11
- l’épitope SEQ ID NO : 50 ou SEQ ID NO : 51 restreint à HLA-A24 - the epitope SEQ ID NO: 50 or SEQ ID NO: 51 restricted to HLA-A24
- l’épitope SEQ ID NO : 55 restreint à HLA-B7 , HLA-B18 et HLA-B51 , - the epitope SEQ ID NO: 55 restricted to HLA-B7, HLA-B18 and HLA-B51,
- l’épitope SEQ ID NO : 52 restreint à HLA-B15 - the epitope SEQ ID NO: 52 restricted to HLA-B15
- l’épitope SEQ ID NO : 56 restreint à HLA-B35 - the epitope SEQ ID NO: 56 restricted to HLA-B35
- l’épitope SEQ ID NO : 54 (restreint à HLA-B40 et HLA-B44, et - the epitope SEQ ID NO: 54 (restricted to HLA-B40 and HLA-B44, and
- l’épitope SEQ ID NO : 59 ou SEQ ID NO : 66 restreint à HLA-B44. - the epitope SEQ ID NO: 59 or SEQ ID NO: 66 restricted to HLA-B44.
[0016] Dans des modes de réalisation particuliers, ladite composition immunogène ou vaccinale comprend en outre au moins un épitope T CD4 de la NP ou de la GP du virus Ebola inclus dans une séquence choisie parmi les séquences SEQ ID NO : 74 à 96 et 129 à 138 de la NP et les séquences SEQ ID NO : 97 à 128 et 139 à 148 de la GP. In particular embodiments, said immunogenic or vaccine composition further comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of the GP.
[0017] Dans des modes de réalisation particuliers de ladite composition immunogène ou vaccinale, ladite combinaison d’épitopes T CD8, et éventuellement les autres épitopes sont inclus dans un ou plusieurs peptides issus de la protéine NP de séquence SEQ ID NO : 1 ou GP de séquence SEQ ID NO : 49, polypeptides multi-épitopiques ou protéines chimériques, et/ou polynucléotides ou vecteurs recombinants codant le ou lesdits peptides, polypeptides et/ou protéines. De préférence, ladite composition immunogène ou vaccinale comprend les peptides NP de séquence SEQ ID NO : 80, 135, 149, 150, 151 et 152 ou les peptides GP de séquence SEQ ID NO : 107, 148, 153, 154, 155 et 156. In particular embodiments of said immunogenic or vaccine composition, said combination of CD8 T epitopes, and optionally the other epitopes are included in one or more peptides derived from the NP protein of sequence SEQ ID NO: 1 or GP of sequence SEQ ID NO: 49, multi-epitope polypeptides or chimeric proteins, and / or polynucleotides or recombinant vectors encoding said peptide (s), polypeptides and / or proteins. Preferably, said immunogenic or vaccine composition comprises the NP peptides of sequence SEQ ID NO: 80, 135, 149, 150, 151 and 152 or the GP peptides of sequence SEQ ID NO: 107, 148, 153, 154, 155 and 156 .
[0018] La présente invention a également pour objet ladite composition immunogène ou vaccinale selon la présente description pour son utilisation comme vaccin dans la prévention d’une infection par le virus Ebola. [0018] A subject of the present invention is also said immunogenic or vaccine composition according to the present description for its use as a vaccine in the prevention of an infection by the Ebola virus.
[0019] La présente invention a également pour objet l’utilisation in vitro de ladite composition immunogène ou vaccinale selon la présente description pour le diagnostic d’une infection par le virus Ebola ou l’immunomonitorage de la réponse cellulaire contre le virus Ebola. Exposé de l’invention A subject of the present invention is also the in vitro use of said immunogenic or vaccine composition according to the present description for the diagnosis of an infection by the Ebola virus or the immunomonitoring of the cellular response against the Ebola virus. Disclosure of the invention
[0020] En conséquence, la présente invention a pour objet une composition immunogène ou vaccinale contre le virus Ebola, comprenant au moins une combinaison d’épitopes T CD8 du virus Ebola qui est apte à être présentée par au moins quatre molécules HLA I prépondérantes différentes et à induire une réponse en lymphocytes T CD8+ humains spécifiques du virus Ebola chez des individus exprimant au moins l’une desdites molécules HLA-I. Consequently, the present invention relates to an immunogenic or vaccine composition against the Ebola virus, comprising at least one combination of CD8 T epitopes of the Ebola virus which is capable of being presented by at least four different predominant HLA I molecules. and inducing a human CD8 + T lymphocyte response specific for Ebola virus in individuals expressing at least one of said HLA-I molecules.
Conformément à la présente invention : In accordance with the present invention:
- Les séquences issues de la nucléoprotéine (NP) du virus Ebola sont définies en référence à la NP de séquence SEQ ID NO : 1 ou UniProt - The sequences derived from the nucleoprotein (NP) of the Ebola virus are defined with reference to the NP of sequence SEQ ID NO: 1 or UniProt
AAD14590.1 , issue de la souche Zaïre du virus Ebola (ZEBOV). AAD14590.1, from the Zaire strain of the Ebola virus (ZEBOV).
- Les séquences issues de la glycoprotéine (GP) du virus Ebola sont définies en référence à la GP de séquence SEQ ID NO : 49 ou UniProt AKG65268.1 , issue de la souche Zaïre du virus Ebola (ZEBOV). - The sequences derived from the glycoprotein (GP) of the Ebola virus are defined with reference to the GP of sequence SEQ ID NO: 49 or UniProt AKG65268.1, derived from the Zaire strain of the Ebola virus (ZEBOV).
- Les acides aminés sont désignés à l’aide du code à une lettre. Les positions des peptides issus de la NP du virus Ebola sont indiquées en référence à la séquence SEQ ID NO : 1. Les positions des peptides issus de la GP du virus Ebola sont indiquées en référence à la séquence SEQ ID NO : 49. - Amino acids are designated using the one-letter code. The positions of the peptides derived from the NP of the Ebola virus are indicated with reference to the sequence SEQ ID NO: 1. The positions of the peptides derived from the GP of the Ebola virus are indicated with reference to the sequence SEQ ID NO: 49.
- On entend par épitope T CD8, un peptide de 9 à 10 acides aminés qui lie au moins une molécule HLA I, de préférence une molécule HLA I prépondérante telle que présentée au [Tableau 1 ], et est ainsi présenté par ladite molécule HLA I et reconnu par des lymphocytes T CD8 humains spécifiques chez des individus portant cette molécule HLA I. The term "CD8 T epitope" means a peptide of 9 to 10 amino acids which binds at least one HLA I molecule, preferably a predominant HLA I molecule as presented in [Table 1], and is thus presented by said HLA I molecule. and recognized by specific human CD8 T lymphocytes in individuals carrying this HLA I molecule.
- On entend par épitope T CD8 restreint à une molécule HLA I, un épitope qui est reconnu par des lymphocytes T CD8 humains spécifiques lorsqu’il est présenté par ladite molécule HLA I. - CD8 T epitope restricted to an HLA I molecule is understood to mean an epitope which is recognized by specific human CD8 T lymphocytes when it is presented by said HLA I molecule.
- On entend par épitope T CD8 immunogène, un épitope T CD8 qui est apte à induire une réponse en lymphocytes T CD8 humains spécifiques chez des individus exprimant au moins ladite molécule HLA I. The term “immunogenic CD8 T epitope” means a CD8 T epitope which is capable of inducing a response in specific human CD8 T lymphocytes in individuals expressing at least said HLA I molecule.
- On entend par « molécule HLA I prépondérante », une molécule HLA I dont la fréquence allélique est supérieure à 5 % dans une population de référence, par exemple l’une des populations caucasiennes ou africaines du [Tableau 1] - On entend par épitope T CD4, un peptide de 11 à 25 acides aminés qui lie au moins une molécule HLA II, en particulier au moins un allèle HLA-DR fréquent dans différentes populations tel que présenté au [Tableau 7], et est reconnu par des lymphocytes T CD4 humains spécifiques chez des individus portant cette molécule HLA II; le peptide comprend une séquence de 9 acides aminés incluant les résidus d’ancrage aux molécules HLA II, flanquée d’au moins 1 acide aminé, de préférence au moins 2 ou 3 acides aminés à chacune de ses extrémités. - The term “predominant HLA I molecule” means an HLA I molecule whose allelic frequency is greater than 5% in a reference population, for example one of the Caucasian or African populations of [Table 1]. - CD4 T epitope is understood to mean a peptide of 11 to 25 amino acids which binds at least one HLA II molecule, in particular at least one HLA-DR allele frequent in different populations as presented in [Table 7], and is recognized by specific human CD4 T lymphocytes in individuals carrying this HLA II molecule; the peptide comprises a sequence of 9 amino acids including the residues for anchoring to the HLA II molecules, flanked by at least 1 amino acid, preferably at least 2 or 3 amino acids at each of its ends.
- On entend par « lymphocytes T CD8 ou T CD4 spécifiques », des lymphocytes T CD8 ou T CD4 spécifiques des séquences de la NP ou de la GP du virus Ebola. Les expressions « lymphocytes T CD8 ou T CD4 spécifiques », « lymphocytes T CD8 spécifiques du virus Ebola », « lymphocytes T CD8 spécifiques de la NP ou de la GP du virus Ebola », « lymphocytes T CD8 dirigés contre le virus Ebola », « lymphocytes T CD8 dirigés contre la NP ou la GP virus Ebola » sont utilisées indifféremment pour désigner des lymphocytes T CD8 ou T CD4 spécifiques des séquences de la NP ou de la GP du virus Ebola. The expression “specific CD8 T lymphocytes or CD4 T lymphocytes” means CD8 T lymphocytes or CD4 T lymphocytes specific for the NP or GP sequences of the Ebola virus. The expressions “specific CD8 or CD4 T lymphocytes”, “CD8 T lymphocytes specific for the Ebola virus”, “CD8 T lymphocytes specific for the NP or the GP of the Ebola virus”, “CD8 T lymphocytes directed against the Ebola virus”, “CD8 T lymphocytes directed against the NP or GP Ebola virus” are used interchangeably to designate CD8 T lymphocytes or CD4 T lymphocytes specific for the sequences of the NP or the GP of the Ebola virus.
- On entend par « induction d’une réponse en lymphocytes T CD8 ou T CD4 spécifiques », la stimulation de lymphocytes T CD8 ou T CD4 spécifiques in vitro ou in vivo. - The term "induction of a response in specific CD8 T lymphocytes or CD4 T lymphocytes" means the stimulation of specific CD8 T lymphocytes or CD4 T lymphocytes in vitro or in vivo.
On entend par fréquence ou taux de répondeurs in vitro à un épitope T CD4 ou T CD8 ou un peptide comprenant un épitope T CD4 ou T CD8 selon la présente invention, le pourcentage d’individus humains d’un groupe de référence pour lesquels des lignées de lymphocytes T CD4 ou T CD8 humains spécifiques dudit épitope ou peptide ont été obtenues. The term “frequency or level of responders in vitro to a T CD4 or T CD8 epitope or a peptide comprising a T CD4 or T CD8 epitope according to the present invention” is understood to mean the percentage of human individuals of a reference group for which the lines human CD4 or CD8 T lymphocytes specific for said epitope or peptide were obtained.
- On entend par intensité de la réponse in vitro à un peptide comprenant un épitope T CD4 selon la présente invention, le pourcentage de lignées de lymphocytes T CD4 spécifiques dudit peptide qui ont été obtenues dans un groupe d’individus humains de référence par rapport à la totalité des lignées de lymphocytes T CD4 spécifiques obtenues avec les différents peptides de la même protéine (NP ou GP). The term “intensity of the response in vitro to a peptide comprising a CD4 T epitope according to the present invention” means the percentage of CD4 T lymphocyte lines specific for said peptide which were obtained in a group of reference human individuals compared to all of the specific CD4 T lymphocyte lines obtained with the different peptides of the same protein (NP or GP).
- On entend par groupe de référence, un ensemble d’individus humains exprimant des molécules HLA I ou HLA II variées, notamment des molécules HLA I variées incluant les allèles HLA-A et HLA-B les plus fréquents dans différentes populations ([Tableau 1] ou des molécules HLA-DR variées incluant les allèles HLA-DR les plus fréquents dans différentes populations ([Tableau 7]). Le groupe de référence inclus les groupes de référence des exemples (HLA I : Figure 1 , [Tableau 2], [Tableau 3] et [Tableau 4]) ; HLA II : [Tableau 8] et [Tableau 9]). Le groupe de référence est considéré comme représentatif d’une population ou d’un ensemble de populations, c’est-à-dire qu’il fournit des résultats extrapolables à cette ou ces population(s). - The term "reference group" is understood to mean a set of human individuals expressing various HLA I or HLA II molecules, in particular various HLA I molecules including the most frequent HLA-A and HLA-B alleles in different populations ([Table 1] or various HLA-DR molecules including the most frequent HLA-DR alleles in different populations ([Table 7]). The reference group includes the reference groups of the examples (HLA I: Figure 1, [Table 2], [Table 3] and [Table 4]); HLA II: [Table 8] and [Table 9]). The reference group is considered to be representative of a population or a set of populations, that is to say it provides results that can be extrapolated to this or these population (s).
- On entend par virus Ebola, n’importe quel isolat du virus Ebola. - Ebola virus is understood to mean any isolate of Ebola virus.
- Le pourcentage d’identité d’une séquence d’acide aminé est défini par le pourcentage de résidus d’acides aminés dans une séquence à comparer qui sont identiques à une séquence de référence après alignement des séquences, en introduisant des espaces si nécessaire, de façon à obtenir une identité de séquence maximale. L’alignement de séquences en vue de déterminer le pourcentage d’identité d’une séquence peut être réalisé de différentes façons connues de l’homme du métier, par exemple en utilisant des logiciels publics disponibles comme BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). Ce logiciel est de préférence utilisé avec des paramètres par défaut. - The percentage identity of an amino acid sequence is defined by the percentage of amino acid residues in a sequence to be compared which are identical to a reference sequence after alignment of the sequences, by introducing spaces if necessary, so as to obtain maximum sequence identity. The alignment of sequences with a view to determining the percentage identity of a sequence can be carried out in various ways known to those skilled in the art, for example using publicly available software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). This software is preferably used with default settings.
- Sauf indication contraire, un, signifie au moins un, et ou signifie et/ou. - Unless otherwise indicated, a, means at least one, and or means and / or.
[0021] La capacité des épitopes T CD8 et peptides dérivés comprenant ces épitopes selon l’invention de lier des molécules HLA I est évaluée selon les techniques standards connues de l’Homme du métier telles que celles décrites en particulier dans les exemples. Il s’agit notamment de méthodes in silico Les méthodes in silico utilisent des outils de prédiction de liaison au CMH-I, tel que par exemple NetMHC v4.0. La capacité des épitopes T CD8 et peptides dérivés de l’invention de stimuler des lymphocytes T CD8 humains spécifiques, par exemple à partir de précurseurs présents chez des individus humains naïfs, de stimuler spécifiquement de telles cellules chez des individus humains ayant été infectés par le virus Ebola, la spécificité des lymphocytes T CD8 induits vis-à-vis des peptides ou de la protéine NP ou GP du virus Ebola, ainsi que la capacité des épitopes T CD8 et peptides dérivés selon l’invention d’être reconnus par des lymphocytes T CD8 spécifiques, est évaluée selon les techniques standards connues de l’Homme du métier telles que celles décrites dans les exemples. Il s’agit notamment d’un test de prolifération cellulaire, d’un test ELISPOT (dosage des cellules productrices de cytokines) ou d’un test de dosage de cytokines intracellulaires, spécifiques d’une cytokine produite par des lymphocytes T CD8 activés, en particulier une cytokine de type Th1 telle que par exemple IFN-g, IL-2 ou TNF-a. Les exemples montrent que les épitopes T CD8 et peptides dérivés selon l’invention sont capables d’induire une réponse en lymphocytes T CD8 humains spécifiques à partir de précurseurs présents chez des individus humains naïfs d’un groupe de référence. The capacity of CD8 T epitopes and derivative peptides comprising these epitopes according to the invention to bind HLA I molecules is evaluated according to standard techniques known to those skilled in the art such as those described in particular in the examples. These are in particular in silico methods The in silico methods use tools for predicting binding to MHC-I, such as for example NetMHC v4.0. The ability of CD8 T epitopes and peptides derived from the invention to stimulate specific human CD8 T lymphocytes, for example from precursors present in naive human individuals, to specifically stimulate such cells in human individuals who have been infected with the Ebola virus, the specificity of the CD8 T lymphocytes induced vis-à-vis the peptides or the NP or GP protein of the Ebola virus, as well as the ability of the CD8 T epitopes and peptides derived according to the invention to be recognized by lymphocytes T CD8 specific, is evaluated according to standard techniques known to those skilled in the art such as those described in the examples. he This concerns in particular a cell proliferation test, an ELISPOT test (assay of cytokine-producing cells) or an assay for assaying intracellular cytokines, specific for a cytokine produced by activated CD8 T lymphocytes, in in particular a Th1 type cytokine such as, for example, IFN-g, IL-2 or TNF-a. The examples show that the CD8 T epitopes and peptides derived according to the invention are capable of inducing a response in specific human CD8 T lymphocytes from precursors present in naive human individuals of a reference group.
[0022] Selon un mode de réalisation de l’invention, lesdites molécules HLA I prépondérantes sont choisies parmi HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA- A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA-B40, HLA- B44 et HLA-B51 ; de préférence parmi HLA-A*01 :01 , HLA-A*02:01 , HLA-A*03:01 , HLA-A*11 :01 , HLA-A*24:02, HLA-A*29:02, HLA-B*07:02, HLA-B*08:01 , HLA-[0022] According to one embodiment of the invention, said predominant HLA I molecules are chosen from HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-A29, HLA-B7, HLA -B8, HLA-B15, HLA-B18, HLA-B35, HLA-B40, HLA-B44 and HLA-B51; preferably from HLA-A * 01: 01, HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11: 01, HLA-A * 24: 02, HLA-A * 29: 02 , HLA-B * 07: 02, HLA-B * 08: 01, HLA-
B*15:01 , HLA-B*15:02, HLA-B*15:03, HLA-B*18:01 , HLA-B*35:01 , HLA-B*35:02, HLA-B*35:03, HLA-B*40:01 , HLA-B*44:02, HLA-B*44:03 et HLA-B*51 :01 ; de manière préférée parmi HLA-A*01 :01 , HLA-A*02:01 , HLA-A*03:01 , HLA-A*11 :01 , HLA-A*24:02, HLA-A*29:02, HLA-B*07:02, HLA-B*08:01 , HLA-B*15:01 , HLA-B * 15:01, HLA-B * 15: 02, HLA-B * 15: 03, HLA-B * 18: 01, HLA-B * 35: 01, HLA-B * 35: 02, HLA-B * 35:03, HLA-B * 40:01, HLA-B * 44:02, HLA-B * 44:03 and HLA-B * 51:01; preferably from among HLA-A * 01: 01, HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11: 01, HLA-A * 24:02, HLA-A * 29: 02, HLA-B * 07:02, HLA-B * 08:01, HLA-B * 15:01, HLA-
B*18:01 , HLA-B*35:01 , HLA-B*35:03, HLA-B*40:01 , HLA-B*44:02, HLA-B*44:03 et HLA-B*51 :01 ; ces allèles HLA-I sont représentatifs des populations Caucasienne et Africaine. En particulier, cet ensemble d’allèles permet de couvrir 95,2 % des individus de la population mondiale ; 98,7 % des individus d’Europe ; 67,9 % des individus d’Afrique Centrale ; 71 ,9 des individus d’Afrique de l’Est ; 80,8 des individus d’Afrique Sub-Saharienne ; 80,7 des individus d’Afrique du Nord et 74,4% des individus d’Afrique de l’Ouest ; 87,9% des individus d’Asie du Nord-Est ; 78,8% des individus d’Asie du Sud ; 88,9% des individus d’Asie du Sud-Est ; 81 ,2% des individus d’Asie du Sud-Ouest ; 92,2% des individus d’Asie de l’Est ; 92,1 % des individus d’Océanie ; 95,1 % des individus d’Amérique du Nord ; 73,4% des individus d’Amérique du Sud et 93,4% des individus des Caraïbes B * 18: 01, HLA-B * 35: 01, HLA-B * 35: 03, HLA-B * 40: 01, HLA-B * 44: 02, HLA-B * 44: 03 and HLA-B * 51:01; these HLA-I alleles are representative of the Caucasian and African populations. In particular, this set of alleles covers 95.2% of individuals in the world population; 98.7% of people in Europe; 67.9% of people from Central Africa; 71.9 individuals from East Africa; 80.8 people from Sub-Saharan Africa; 80.7 of individuals from North Africa and 74.4% of individuals from West Africa; 87.9% of individuals from Northeast Asia; 78.8% of individuals from South Asia; 88.9% of individuals from Southeast Asia; 81.2% of individuals from Southwest Asia; 92.2% of individuals from East Asia; 92.1% of people from Oceania; 95.1% of people from North America; 73.4% of people from South America and 93.4% of people from the Caribbean
[0023] Selon un mode de réalisation de l’invention, lesdits épitopes CD8 sont sélectionnés parmi les séquences suivantes de la nucléoprotéine (NP) et/ou de la glycoprotéine (GP) du virus Ebola : - SEQ ID NO : 28 et 46 de la NP, restreints à HLA-A1 [0023] According to one embodiment of the invention, said CD8 epitopes are selected from the following sequences of the nucleoprotein (NP) and / or of the glycoprotein (GP) of the Ebola virus: - SEQ ID NO: 28 and 46 of the NP, restricted to HLA-A1
- SEQ ID NO : 2, 5, 7, 8, 1 1 , 13, 14, 17, 20, 23, 36, 38 et 45 de la NP et SEQ ID - SEQ ID NO: 2, 5, 7, 8, 11, 13, 14, 17, 20, 23, 36, 38 and 45 of the NP and SEQ ID
NO : 53, 57, 58, 63 , 64, 65, 72 et 73 de la GP, restreints à HLA-A2 NO: 53, 57, 58, 63, 64, 65, 72 and 73 of the GP, restricted to HLA-A2
- SEQ ID NO : 9 et 44 de la NP et SEQ ID NO : 69 de la GP, restreints à HLA-A3 - SEQ ID NO: 9 and 44 of the NP and SEQ ID NO: 69 of the GP, restricted to HLA-A3
- SEQ ID NO : 24 de la NP et SEQ ID NO : 71 de la GP, restreints à HLA-A1 1- SEQ ID NO: 24 of the NP and SEQ ID NO: 71 of the GP, restricted to HLA-A1 1
- SEQ ID NO : 16 de la NP et SEQ ID NO : 50 et 51 de la GP restreints à HLA- A24 - SEQ ID NO: 16 of the NP and SEQ ID NO: 50 and 51 of the GP restricted to HLA-A24
- SEQ ID NO : 10 et 41 de la NP, restreints à HLA-A29 - SEQ ID NO: 10 and 41 of the NP, restricted to HLA-A29
- SEQ ID NO : 33 de la NP, restreint à HLA-B7 - NP SEQ ID NO: 33, restricted to HLA-B7
- SEQ ID NO : 26 de la NP, restreint à HLA-B8 - NP SEQ ID NO: 26, restricted to HLA-B8
- SEQ ID NO : 12, 15, 22, 29 et 37 de la NP et SEQ ID NO : 52 de la GP, restreints à HLA-B15 - SEQ ID NO: 12, 15, 22, 29 and 37 of the NP and SEQ ID NO: 52 of the GP, restricted to HLA-B15
- SEQ ID NO : 25 de la NP, restreint à HLA-B18 - NP SEQ ID NO: 25, restricted to HLA-B18
- SEQ ID NO : 19, 40, 43, 48 de la NP et SEQ ID NO : 56 de la GP, restreints à - SEQ ID NO: 19, 40, 43, 48 of the NP and SEQ ID NO: 56 of the GP, restricted to
HLA-B35 HLA-B35
- SEQ ID NO : 3, 30, 39 et 47 de la NP et SEQ ID NO : 60 et 68 de la GP, restreints à HLA-B40 - SEQ ID NO: 3, 30, 39 and 47 of the NP and SEQ ID NO: 60 and 68 of the GP, restricted to HLA-B40
- SEQ ID NO : 4 et 31 de la NP et SEQ ID NO : 59, 62, 66 et 67 de la GP, restreints à HLA-B44 - SEQ ID NO: 4 and 31 of the NP and SEQ ID NO: 59, 62, 66 and 67 of the GP, restricted to HLA-B44
- SEQ ID NO : 18 et 32 de la NP et SEQ ID NO : 70 de la GP, restreints à HLA- B51 - SEQ ID NO: 18 and 32 of the NP and SEQ ID NO: 70 of the GP, restricted to HLA-B51
- SEQ ID NO : 61 de la GP, restreint à HLA-A1 et HLA-A29 - GP SEQ ID NO: 61, restricted to HLA-A1 and HLA-A29
- SEQ ID NO : 21 de la NP, restreint à HLA-A2 et HLA-A24 - NP SEQ ID NO: 21, restricted to HLA-A2 and HLA-A24
- SEQ ID NO : 6 de la NP, restreint à HLA-A29 et HLA-B15 - NP SEQ ID NO: 6, restricted to HLA-A29 and HLA-B15
- SEQ ID NO : 35 de la NP, restreint à HLA-B7 et HLA-B35 - NP SEQ ID NO: 35, restricted to HLA-B7 and HLA-B35
- SEQ ID NO : 27 de la NP, restreint à HLA-B7 et HLA-B51 - NP SEQ ID NO: 27, restricted to HLA-B7 and HLA-B51
- SEQ ID NO : 55 de la GP, restreint à HLA-B7, HLA-B18 et HLA-B51 - GP SEQ ID NO: 55, restricted to HLA-B7, HLA-B18 and HLA-B51
- SEQ ID NO : 34 et 42 de la NP, restreints à HLA-B18 et HLA-B40, et - SEQ ID NO: 34 and 42 of NP, restricted to HLA-B18 and HLA-B40, and
- SEQ ID NO : 54 de la GP, restreint à HLA-B40 et HLA-B44. - SEQ ID NO: 54 of the GP, restricted to HLA-B40 and HLA-B44.
[0024] Les séquences ci-dessus sont issues du virus Ebola Zaïre. Toutefois, l’invention englobe les épitopes variants naturels ou synthétiques obtenus par mutation (insertion, délétion, substitution) d’un ou plusieurs acides aminés dans la séquence de la nucléoprotéine de séquence SEQ ID NO : 1 ou de la glycoprotéine de séquence SEQ ID NO : 49, dès lors que ledit peptide conserve ses propriétés d’épitope T CD8 immunogène du virus Ebola telles que définies ci-dessus, c’est-à- dire qu’il est reconnu par des lymphocytes T CD8 humains spécifiques de la séquence sauvage ou d’un variant naturel du virus Ebola. Les épitopes variants comprennent avantageusement au plus 3 mutations dans l’une des séquences SEQ ID NO : 2 à 48 et 50 à 73, de préférence au plus 3 substitutions dans lesdites séquences. [0024] The above sequences are from the Zaire Ebola virus. However, the invention encompasses the natural or synthetic variant epitopes obtained by mutation (insertion, deletion, substitution) of one or more amino acids in the sequence of the nucleoprotein of sequence SEQ ID NO: 1 or of the glycoprotein of sequence SEQ ID NO: 49, since said peptide retains its properties of the immunogenic CD8 T epitope of the Ebola virus as defined above, it is that is, it is recognized by human CD8 T lymphocytes specific for the wild-type sequence or for a natural variant of the Ebola virus. The variant epitopes advantageously comprise at most 3 mutations in one of the sequences SEQ ID NO: 2 to 48 and 50 to 73, preferably at most 3 substitutions in said sequences.
[0025] Ladite combinaison d’épitopes comprend avantageusement 5 ou plus épitopes T CD8 selon l’invention, par exemple 5 à 15 (5, 6, 7, 8, 9, 10, 1 1 , 12, 13, [0025] Said combination of epitopes advantageously comprises 5 or more CD8 T epitopes according to the invention, for example 5 to 15 (5, 6, 7, 8, 9, 10, 1 1, 12, 13,
14, 15), 5 à 25 (5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23,14, 15), 5 to 25 (5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25), 10 à 25 (10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25) ou plus épitopes T CD8 selon l’invention. La combinaison d’épitopes de la composition est choisie de manière à ce qu’elle soit apte à être présentée par au moins 4 molécules HLA I prépondérantes différentes, de préférence au moins 8 (8, 9, 10, 11 , 12, 13 ou 14) molécules HLA I prépondérantes différentes, de manière préférée au moins 12 (12, 13 ou 14) molécules HLA I prépondérantes différentes, de manière encore plus préférée 13 ou la totalité desdites molécules HLA I prépondérantes. 24, 25), 10 to 25 (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) or more CD8 T epitopes according to the invention . The combination of epitopes of the composition is chosen so that it is capable of being presented by at least 4 different predominant HLA I molecules, preferably at least 8 (8, 9, 10, 11, 12, 13 or 14) different preponderant HLA I molecules, preferably at least 12 (12, 13 or 14) different preponderant HLA I molecules, even more preferably 13 or all of said preponderant HLA I molecules.
[0026] Ladite composition comprenant une combinaison d’épitopes T CD8 de la NP et/ou de la GP du virus Ebola telle que définie précédemment permet d’améliorer la réponse immunitaire contre le virus Ebola en élargissant la couverture d’individus humains répondeurs. [0026] Said composition comprising a combination of CD8 T epitopes of the NP and / or of the GP of the Ebola virus as defined above makes it possible to improve the immune response against the Ebola virus by broadening the coverage of responder human individuals.
[0027] Selon un mode de réalisation avantageux de l’invention ladite combinaison d’épitopes comprend au moins un épitope sélectionné dans le groupe constitué par les séquences SEQ ID NO : 6, 21 , 27, 34, 35, 42 de la NP et SEQ ID NO : 54, 55 et 61 de la GP ; ces épitopes sont restreints à plusieurs molécules HLA I permettant ainsi de limiter le nombre de séquences peptidiques dans la composition. According to an advantageous embodiment of the invention, said combination of epitopes comprises at least one epitope selected from the group consisting of the sequences SEQ ID NO: 6, 21, 27, 34, 35, 42 of the NP and GP SEQ ID NO: 54, 55 and 61; these epitopes are restricted to several HLA I molecules, thus making it possible to limit the number of peptide sequences in the composition.
[0028] Selon un mode de réalisation avantageux de l’invention, les épitopes de la combinaison sont sélectionnés dans le groupe constitué par : les séquences SEQ ID NO : 3 à 7, 9 à 22, 24 à 35 et 37 à 48 de la NP et les séquences SEQ ID NO : 50 à 52, 54 à 56 et 59 à 64 et 66 à 71 de la GP. Ces épitopes induisent avantageusement une fréquence de répondeurs d’au moins 25 % chez les individus portant l’allèle ou l’un des allèles HLA I auquel l’épitope est restreint tels que définis ci-dessus ([Tableau 2], [Tableau 3] et [Tableau 4]). According to an advantageous embodiment of the invention, the epitopes of the combination are selected from the group consisting of: the sequences SEQ ID NO: 3 to 7, 9 to 22, 24 to 35 and 37 to 48 of the NP and the sequences SEQ ID NO: 50 to 52, 54 to 56 and 59 to 64 and 66 to 71 of the GP. These epitopes advantageously induce a responder frequency of at least 25% in individuals carrying the allele or one of the HLA I alleles to which the epitope is restricted as defined above ([Table 2], [Table 3 ] and [Table 4]).
[0029] De préférence, l’un au moins des épitopes de la combinaison est sélectionné dans le groupe constitué par : les séquences SEQ ID NO : 5 à 7, 10 à 21 (HLA-A2), 22, 24 à 35, 37 à 45 et 48 de la NP et les séquences SEQ ID NO : 52, 54 (HLA-B40), 55, 56, 59, 61 (HLA-A29), 63, 64, 66 et 68 à 71 de la GP. Ces épitopes induisent avantageusement une fréquence de répondeurs d’au moins 33,3 % chez les individus portant l’allèle ou l’un des allèles HLA I auquel l’épitope est restreint tels que définis ci-dessus ou l’allèle HLA I tel qu’indiqué entre parenthèses ([Tableau 2], [Tableau 3] et [Tableau 4]). Preferably, at least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5 to 7, 10 to 21 (HLA-A2), 22, 24 to 35, 37 to 45 and 48 of the NP and the sequences SEQ ID NO: 52, 54 (HLA-B40), 55, 56, 59, 61 (HLA-A29), 63, 64, 66 and 68 to 71 of the GP. These epitopes advantageously induce a responder frequency of at least 33.3% in individuals carrying the allele or one of the HLA I alleles to which the epitope is restricted as defined above or the HLA I allele such as as indicated in parentheses ([Table 2], [Table 3] and [Table 4]).
[0030] De manière préférée, l’un au moins des épitopes de la combinaison est sélectionné dans le groupe constitué par : les séquences SEQ ID NO : 5, 6, 10, 12, 14 à 17, 20, 22, 24 à 27(HLA-B7), 28 à 31 , 33, 34, 35 (HLA-B35), 37, 39 à 42, 44, 45 de la NP et les séquences SEQ ID NO : 52, 54 (HLA-B40), 55 (HLA-B18), 59, 61 (HLA-A29), 63, 64, 66, 68, 69 et 71 de la GP. Ces épitopes induisent avantageusement une fréquence de répondeurs d’au moins 50 % chez les individus portant l’allèle ou l’un des allèles HLA I tels que définis ci-dessus ou l’allèle HLA I tel qu’indiqué entre parenthèses ([Tableau 2], [Tableau 3] et [Tableau 4])· Preferably, at least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 12, 14 to 17, 20, 22, 24 to 27 (HLA-B7), 28 to 31, 33, 34, 35 (HLA-B35), 37, 39 to 42, 44, 45 of the NP and the sequences SEQ ID NO: 52, 54 (HLA-B40), 55 (HLA-B18), 59, 61 (HLA-A29), 63, 64, 66, 68, 69 and 71 of the GP. These epitopes advantageously induce a responder frequency of at least 50% in individuals carrying the allele or one of the HLA I alleles as defined above or the HLA I allele as indicated in parentheses ([Table 2], [Table 3] and [Table 4]) ·
[0031] De manière encore plus préférée, l’un au moins des épitopes de la combinaison est sélectionné dans le groupe constitué par : les séquences SEQ ID NO : 5, 6, 10, 15, 17, 24, 25, 34, 41 et 42 de la NP et les séquences SEQ ID NO : 55 (HLA-B18), 61 (HLA-A29) et 71 de la GP. Ces épitopes induisent avantageusement une fréquence de répondeurs de 75 % à 100 % chez les individus portant l’allèle ou l’un des allèles HLA I tels que définis ci-dessus ou l’allèle HLA I tel qu’indiqué entre parenthèses ([Tableau 2], [Tableau 3] et [Tableau 4])· Even more preferably, at least one of the epitopes of the combination is selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 15, 17, 24, 25, 34, 41 and 42 of NP and the sequences SEQ ID NO: 55 (HLA-B18), 61 (HLA-A29) and 71 of GP. These epitopes advantageously induce a responder frequency of 75% to 100% in individuals carrying the allele or one of the HLA I alleles as defined above or the HLA I allele as indicated in parentheses ([Table 2], [Table 3] and [Table 4]) ·
[0032] Selon un mode de réalisation avantageux de l’invention, la dite combinaison comprend au moins un épitope T CD8 conservé, c’est-à-dire dont la séquence est conservée dans au moins une autre espèce du virus Ebola, de préférence Ebola Soudan ; de manière préférée une séquence conservée dans Ebola Zaïre Soudan, Reston, Bundibugyo et Tai Forest. De préférence, la séquence dudit peptide présente au moins 66%, de préférence au moins 75%, 80%, 85%, 90 %, 95 %, 98 %, 99% ou 100 % d’identité avec la séquence d’une autre espèce du virus Ebola, de préférence Ebola Soudan ; de manière préférée avec les séquences des virus Ebola Soudan, Reston, Bundibugyo et Tai Forest. La composition comprend avantageusement au moins un peptide NP choisi parmi les séquences SEQ ID NO : 3 à 8, 10 à 39, 41 , 43, 47 et 48 qui présentent au moins 66% d’identité avec la séquence d’Ebola Soudan; de préférence choisi parmi les séquences SEQ ID NO : 5, 6, 12, 15 à 24, 27 à 38 et 41 qui présentent au moins 85% d’identité avec la séquence d’Ebola Soudan ; de manière préférée choisi parmi les séquences SEQ ID NO : 15 à 18, 20 à 24, 27, 30 à 33, 36 et 37 qui présentent au moins 90% d’identité avec les séquences d’Ebola Soudan, Reston, Bundibugyo et Tai Forest ou au moins un peptide GP choisi parmi les séquences SEQ ID NO : 51 à 60 et 67 à 71 qui présentent au moins 66% d’identité avec la séquence d’Ebola Soudan; de préférence choisi parmi les séquences SEQ ID NO : 51 , 54, 55, 57 à 60, 67 et 70 qui présentent au moins 85% d’identité avec la séquence d’Ebola Soudan ; de manière préférée choisi parmi les séquences SEQ ID NO : 55, 57, 58, 60 et 67 qui présentent au moins 90% d’identité avec les séquences d’Ebola Soudan, Reston, Bundibugyo et Tai Forest. According to an advantageous embodiment of the invention, said combination comprises at least one conserved CD8 T epitope, that is to say the sequence of which is conserved in at least one other species of the Ebola virus, of preference Ebola Sudan; preferably a sequence conserved in Ebola Zaire Sudan, Reston, Bundibugyo and Tai Forest. Preferably, the sequence of said peptide has at least 66%, preferably at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the sequence of another Ebola virus species, preferably Ebola Sudan; preferably with the sequences of the Ebola Sudan, Reston, Bundibugyo and Tai Forest viruses. The composition advantageously comprises at least one NP peptide chosen from the sequences SEQ ID NO: 3 to 8, 10 to 39, 41, 43, 47 and 48 which have at least 66% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 5, 6, 12, 15 to 24, 27 to 38 and 41 which have at least 85% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 15 to 18, 20 to 24, 27, 30 to 33, 36 and 37 which have at least 90% identity with the sequences of Ebola Sudan, Reston, Bundibugyo and Tai Forest or at least one GP peptide chosen from the sequences SEQ ID NO: 51 to 60 and 67 to 71 which have at least 66% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 51, 54, 55, 57 to 60, 67 and 70 which have at least 85% identity with the sequence of Ebola Sudan; preferably chosen from the sequences SEQ ID NO: 55, 57, 58, 60 and 67 which exhibit at least 90% identity with the sequences of Ebola Sudan, Reston, Bundibugyo and Tai Forest.
[0033] Conformément à l’invention, ladite combinaison comprend des épitopes T CD8 de la NP, des épitopes T CD8 de la GP ou un mélange d’épitopes TCD8 de la NP et de la GP. Selon un mode de réalisation de l’invention, ladite combinaison comprend des épitopes T CD8 de la NP ou un mélange d’épitopes TCD8 de la NP et de la GP. [0033] According to the invention, said combination comprises CD8 T epitopes from NP, CD8 T epitopes from GP or a mixture of TCD8 epitopes from NP and GP. According to one embodiment of the invention, said combination comprises CD8 T epitopes of NP or a mixture of TCD8 epitopes of NP and GP.
[0034] Selon un mode de réalisation préféré de l’invention, ladite composition comprend une combinaison d’épitopes T CD8 de la NP du virus Ebola qui est apte à être présentée par la totalité desdites molécules HLA I et à induire une fréquence maximale de répondeurs in vitro, c’est-à-dire chez tous les individus d’un groupe de référence qui sont répondeurs à la NP (correspondant à 92 % des individus dans le groupe de référence des exemples ; voir [Tableau 2] et [Tableau 3]), ladite combinaison comprenant According to a preferred embodiment of the invention, said composition comprises a combination of CD8 T epitopes of the NP of the Ebola virus which is capable of being presented by all of said HLA I molecules and of inducing a maximum frequency of responders in vitro, that is to say in all the individuals of a reference group who are responders to PN (corresponding to 92% of the individuals in the reference group of the examples; see [Table 2] and [Table 3]), said combination comprising
- au moins les épitopes suivants: - at least the following epitopes:
a) les épitopes SEQ ID NO : 5 restreint à HLA-A2 ; SEQ ID NO : 16 restreint à HLA-A24, SEQ ID NO : 26 restreint à HLA-B8, SEQ ID NO : 28 restreint à HLA-A1 et SEQ ID NO : 31 restreint à HLA-B44 ; a) the epitopes SEQ ID NO: 5 restricted to HLA-A2; SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 28 restricted to HLA-A1 and SEQ ID NO: 31 restricted to HLA-B44;
b) l’un des épitopes SEQ ID NO : 13, SEQ ID NO : 14 ou SEQ ID NO : 17 restreint à HLA-A2 , de préférence l’épitope SEQ ID NO : 17; b) one of the epitopes SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 17 restricted to HLA-A2, preferably the epitope SEQ ID NO: 17;
c) l’un des épitopes SEQ ID NO : 9 ou SEQ ID NO : 44 restreint à HLA- A3 ; de préférence l’épitope SEQ ID NO : 44 ; c) one of the epitopes SEQ ID NO: 9 or SEQ ID NO: 44 restricted to HLA-A3; preferably the epitope SEQ ID NO: 44;
d) l’épitope SEQ ID NO : 24 restreint à HLA-A1 1 ; d) the epitope SEQ ID NO: 24 restricted to HLA-A1 1;
e) l’épitope SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15 ou l’un des épitopes SEQ ID NO : 10 ou SEQ ID NO : 41 restreint à HLA-A29 ; de préférence l’épitope SEQ ID NO : 6; e) the epitope SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15 or one of the epitopes SEQ ID NO: 10 or SEQ ID NO: 41 restricted to HLA-A29; preferably the epitope SEQ ID NO: 6;
f) l’un des épitopes SEQ ID NO : 3, SEQ ID NO : 30, SEQ ID NO : 39 ou SEQ ID NO : 47 restreint à HLA-B40 ou l’un des épitopes SEQ ID NO : 34 ou f) one of the epitopes SEQ ID NO: 3, SEQ ID NO: 30, SEQ ID NO: 39 or SEQ ID NO: 47 restricted to HLA-B40 or one of the epitopes SEQ ID NO: 34 or
SEQ ID NO : 42 restreints à HLA-B40 et HLA-B18, de préférence l’épitope SEQ ID NO : 30, SEQ ID NO : 34 ou SEQ ID NO : 42, de manière plus préférée l’épitope SEQ ID NO : 34 ou SEQ ID NO : 42 et ; SEQ ID NO: 42 restricted to HLA-B40 and HLA-B18, preferably the epitope SEQ ID NO: 30, SEQ ID NO: 34 or SEQ ID NO: 42, more preferably the epitope SEQ ID NO: 34 or SEQ ID NO: 42 and;
g) l’épitope SEQ ID NO : 27 restreint à HLA-B7 et HLA-B51 , l’épitope SEQ ID NO : 33 restreint à HLA-B7 ou l’épitope SEQ ID NO : 35 restreint à HLA-B7 et g) the epitope SEQ ID NO: 27 restricted to HLA-B7 and HLA-B51, the epitope SEQ ID NO: 33 restricted to HLA-B7 or the epitope SEQ ID NO: 35 restricted to HLA-B7 and
HLA-B35 , de préférence l’épitope SEQ ID NO : 27 ou SEQ ID NO : 35 ; de manière plus préférée l’épitope SEQ ID NO : 27 ; HLA-B35, preferably the epitope SEQ ID NO: 27 or SEQ ID NO: 35; more preferably the epitope SEQ ID NO: 27;
- et éventuellement - and eventually
h) l’un des épitopes SEQ ID NO : 18 ou SEQ ID NO : 32 restreint à HLA- B51 ; h) one of the epitopes SEQ ID NO: 18 or SEQ ID NO: 32 restricted to HLA-B51;
i) l’un des épitopes SEQ ID NO : 19, SEQ ID NO : 40, SEQ ID NO : 43, SEQ ID NO : 48 restreint à HLA-B35 ; i) one of the epitopes SEQ ID NO: 19, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 48 restricted to HLA-B35;
j) l’un des épitopes SEQ ID NO : 12, SEQ ID NO : 15, SEQ ID NO : 22, SEQ ID NO : 29 ou SEQ ID NO : 37 restreint à HLA-B15 , de préférence l’épitope SEQ ID NO : 15; j) one of the epitopes SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 29 or SEQ ID NO: 37 restricted to HLA-B15, preferably the epitope SEQ ID NO : 15;
k) l’épitope SEQ ID NO : 25 restreint à HLA-B18 ; k) the epitope SEQ ID NO: 25 restricted to HLA-B18;
L) l’épitope SEQ ID NO : 46 restreint à HLA-A1 ; L) the epitope SEQ ID NO: 46 restricted to HLA-A1;
m) l’épitope SEQ ID NO : 21 restreint à HLA-A24 et HLA-A2 ; n) )l’épitope SEQ ID NO : 4 restreint à HLA-B44 ; et/ou o) l’un des épitopes SEQ ID NO : 2, SEQ ID NO : 7, SEQ ID NO : 8, SEQ ID NO : 1 1 , SEQ ID NO : 20, SEQ ID NO : 23, SEQ ID NO : 36 ou SEQ ID NO : 38 restreints à HLA-A2, de préférence l’épitope SEQ ID NO : 7, SEQ ID NO : 1 1 , SEQ ID NO : 20 ou SEQ ID NO : 38; de manière préférée, l’épitope SEQ ID NO : 38. m) the epitope SEQ ID NO: 21 restricted to HLA-A24 and HLA-A2; n)) the epitope SEQ ID NO: 4 restricted to HLA-B44; and / or o) one of the epitopes SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 1 1, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO : 36 or SEQ ID NO: 38 restricted to HLA-A2, preferably the epitope SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 20 or SEQ ID NO: 38; preferably, the epitope SEQ ID NO: 38.
[0035] De préférence, ladite composition comprend au moins les épitopes SEQ ID NO : 5 restreint à HLA-A2 ; SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15, SEQ ID NO : 16 restreint à HLA-A24, SEQ ID NO : 17 restreint à HLA-A2, SEQ ID NO : 24 restreint à HLA-A1 1 , SEQ ID NO : 26 restreint à HLA-B8, SEQ ID NO : 27 restreint à HLA-B7 et HLA-B51 , SEQ ID NO : 28 restreint à HLA-A1 , SEQ ID NO : 31 restreint à HLA-B44, SEQ ID NO : 34 ou SEQ ID NO : 42 restreints à HLA-B40 et HLA-B18 et SEQ ID NO : 44 restreint à HLA-A3. [0035] Preferably, said composition comprises at least the epitopes SEQ ID NO: 5 restricted to HLA-A2; SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 17 restricted to HLA-A2, SEQ ID NO: 24 restricted to HLA-A1 1, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 27 restricted to HLA-B7 and HLA-B51, SEQ ID NO: 28 restricted to HLA-A1, SEQ ID NO: 31 restricted to HLA-B44, SEQ ID NO : 34 or SEQ ID NO: 42 restricted to HLA-B40 and HLA-B18 and SEQ ID NO: 44 restricted to HLA-A3.
[0036] Par exemple, la dite composition comprend au moins les épitopes SEQ ID NO : 5, SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15, SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15, SEQ ID NO : 9 restreint à HLA-A3, SEQ ID NO : 10 restreint à HLA-A29, SEQ ID NO : 15 restreint à HLA-B15, SEQ ID NO : 16 restreint à HLA-A24, SEQ ID NO : 17 restreint à HLA-A2, SEQ ID NO : 18 restreint à HLA-B51 , SEQ ID NO : 20 restreint à HLA-A2, SEQ ID NO : 21 restreint à HLA-A2 et HLA-A24, SEQ ID NO : 22 restreint à HLA-B15, SEQ ID NO : 24 restreint à HLA-A1 1 , SEQ ID NO : 25 restreint à HLA-B18, SEQ ID NO : 26 restreint à HLA-B8, SEQ ID NO : 27 restreint à HLA-B7 et HLA-B51 , SEQ ID NO : 28 restreint à HLA-A1 , SEQ ID NO : 29 restreint à HLA-B15, SEQ ID NO : 30 restreint à HLA-B40, SEQ ID NO : 31 restreint à HLA-B44, SEQ ID NO : 42 restreints à HLA-B40 et HLA-B18, SEQ ID NO : 43 restreints à HLA-B35, et SEQ ID NO : 44 restreint à HLA-A3. La composition comprenant ladite combinaison d’épitopes T CD8 de la NP du virus Ebola est apte à être présentée par la totalité desdites molécules HLA I et à induire une fréquence maximale de répondeurs in vitro, c’est-à-dire chez tous les individus d’un groupe de référence qui sont répondeurs à la NP (correspondant à 92 % des individus dans le groupe de référence des exemples ; voir [Tableau 12]). [0037] Selon un autre mode de réalisation préféré de l’invention, ladite composition comprend une combinaison d’épitopes T CD8 de la GP du virus Ebola qui est apte à être présentée par 13 desdites molécules HLA I prépondérantes (toutes les molécules HLA I sélectionnées à l’exception de HLA- B8) et à induire une fréquence maximale de répondeurs in vitro, c’est-à-dire chez tous les individus d’un groupe de référence qui sont répondeurs à la GP (correspondant à 60 % des individus dans le groupe de référence des exemples ; voir [Tableau 4]), ladite combinaison comprenant au moins les épitopes suivants: For example, said composition comprises at least the epitopes SEQ ID NO: 5, SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15, SEQ ID NO: 9 restricted to HLA-A3, SEQ ID NO: 10 restricted to HLA-A29, SEQ ID NO: 15 restricted to HLA-B15, SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 17 restricted to HLA-A2, SEQ ID NO: 18 restricted to HLA-B51, SEQ ID NO: 20 restricted to HLA-A2, SEQ ID NO: 21 restricted to HLA-A2 and HLA-A24, SEQ ID NO: 22 restricted to HLA -B15, SEQ ID NO: 24 restricted to HLA-A1 1, SEQ ID NO: 25 restricted to HLA-B18, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 27 restricted to HLA-B7 and HLA- B51, SEQ ID NO: 28 restricted to HLA-A1, SEQ ID NO: 29 restricted to HLA-B15, SEQ ID NO: 30 restricted to HLA-B40, SEQ ID NO: 31 restricted to HLA-B44, SEQ ID NO: 42 restricted to HLA-B40 and HLA-B18, SEQ ID NO: 43 restricted to HLA-B35, and SEQ ID NO: 44 restricted to HLA-A3. The composition comprising said combination of CD8 T epitopes of Ebola virus NP is capable of being presented by all of said HLA I molecules and of inducing a maximum frequency of responders in vitro, that is to say in all individuals. of a reference group who are responders to PN (corresponding to 92% of individuals in the reference group of the examples; see [Table 12]). [0037] According to another preferred embodiment of the invention, said composition comprises a combination of CD8 T epitopes of the Ebola virus GP which is capable of being presented by 13 of said predominant HLA I molecules (all HLA I molecules selected with the exception of HLA-B8) and to induce a maximum frequency of responders in vitro, that is to say in all the individuals of a reference group who are responders to GP (corresponding to 60% of individuals in the reference group of the examples; see [Table 4]), said combination comprising at least the following epitopes:
- l’épitope SEQ ID NO : 61 , restreint à HLA-A1 et HLA-A29 ; - the epitope SEQ ID NO: 61, restricted to HLA-A1 and HLA-A29;
- l’épitope SEQ ID NO : 63 ou SEQ ID NO : 64, restreint à HLA-A2 ; - the epitope SEQ ID NO: 63 or SEQ ID NO: 64, restricted to HLA-A2;
- l’épitope SEQ ID NO : 69, restreint à HLA-A3 ; - the epitope SEQ ID NO: 69, restricted to HLA-A3;
- l’épitope SEQ ID NO : 71 , restreint à HLA-A11 ; - the epitope SEQ ID NO: 71, restricted to HLA-A11;
- l’épitope SEQ ID NO : 50 ou SEQ ID NO : 51 , restreint à HLA-A24 ; - the epitope SEQ ID NO: 50 or SEQ ID NO: 51, restricted to HLA-A24;
-l’épitope SEQ ID NO : 55, restreint à HLA-B7 , HLA-B18 et HLA-B5 ; , the epitope SEQ ID NO: 55, restricted to HLA-B7, HLA-B18 and HLA-B5; ,
- l’épitope SEQ ID NO : 52, restreint à HLA-B15 ; - the epitope SEQ ID NO: 52, restricted to HLA-B15;
- l’épitope SEQ ID NO : 56, restreint à HLA-B35 ; - the epitope SEQ ID NO: 56, restricted to HLA-B35;
- l’épitope SEQ ID NO : 54 restreint à HLA-B40 et HLA-B44 ; et - the epitope SEQ ID NO: 54 restricted to HLA-B40 and HLA-B44; and
- l’épitope SEQ ID NO : 59 ou SEQ ID NO : 66, restreint à HLA-B44. - the epitope SEQ ID NO: 59 or SEQ ID NO: 66, restricted to HLA-B44.
[0038] De préférence, ladite composition comprend au moins les épitopes SEQ ID NO : 51 restreint à HLA-A24 ; SEQ ID NO : 52 restreint à HLA-B15, SEQ ID NO : 53 restreint à HLA-A2, SEQ ID NO : 54 restreint à HLA-B40 et HLA-B44, SEQ ID NO : 55 restreint à HLA-B7, HLA-B18 et HLA-B51 , SEQ ID NO : 56 restreint à HLA-B35, SEQ ID NO : 59 restreint à HLA-B44, SEQ ID NO : 60 restreint à HLA-B44, SEQ ID NO : 61 , restreint à HLA-A1 et HLA-A29, SEQ ID NO : 62 restreint à HLA-B44, SEQ ID NO : 63 restreint à HLA-A2, SEQ ID NO : 64 restreint à HLA-A2, SEQ ID NO : 67 restreint à HLA-B44, SEQ ID NO : 68 restreint à HLA-B40 et SEQ ID NO : 69 restreint à HLA-A3. La composition comprenant ladite combinaison d’épitopes T CD8 de la GP du virus Ebola est apte à être présentée par 13 desdites molécules HLA I prépondérantes (toutes les molécules HLA I sélectionnées à l’exception de HLA-B8) et à induire une fréquence maximale de répondeurs in vitro, c’est-à-dire chez tous les individus d’un groupe de référence qui sont répondeurs à la GP (correspondant à 60 % des individus dans le groupe de référence des exemples ; voir [Tableau 13]) Preferably, said composition comprises at least the epitopes SEQ ID NO: 51 restricted to HLA-A24; SEQ ID NO: 52 restricted to HLA-B15, SEQ ID NO: 53 restricted to HLA-A2, SEQ ID NO: 54 restricted to HLA-B40 and HLA-B44, SEQ ID NO: 55 restricted to HLA-B7, HLA- B18 and HLA-B51, SEQ ID NO: 56 restricted to HLA-B35, SEQ ID NO: 59 restricted to HLA-B44, SEQ ID NO: 60 restricted to HLA-B44, SEQ ID NO: 61, restricted to HLA-A1 and HLA-A29, SEQ ID NO: 62 restricted to HLA-B44, SEQ ID NO: 63 restricted to HLA-A2, SEQ ID NO: 64 restricted to HLA-A2, SEQ ID NO: 67 restricted to HLA-B44, SEQ ID NO: 68 restricted to HLA-B40 and SEQ ID NO: 69 restricted to HLA-A3. The composition comprising said combination of CD8 T epitopes of the Ebola virus GP is capable of being presented by 13 of said predominant HLA I molecules (all the HLA I molecules selected except for HLA-B8) and of inducing a maximum frequency responders in vitro, i.e. in all individuals of a reference group who are responders to GP (corresponding to 60% of individuals in the reference group of the examples; see [Table 13])
[0039] Ces exemples qui sont donnés uniquement à titre illustratif ne sont pas limitatifs et d’autres compositions induisant des fréquences maximales de réponse équivalentes peuvent être aisément obtenues avec d’autres combinaisons d’épitopes déterminées à partir des profils de réponse aux peptides NP et GP du groupe de référence des exemples présentées au [Tableau 2] et au [Tableau 3] pour la NP et au [Tableau 4] pour la GP. These examples, which are given solely by way of illustration, are not limiting and other compositions inducing equivalent maximum response frequencies can be easily obtained with other combinations of epitopes determined from the response profiles to the NP peptides and GP of the reference group of the examples shown in [Table 2] and [Table 3] for PN and [Table 4] for GP.
[0040] Selon un mode de réalisation de l’invention, ladite composition comprend au moins un autre épitope, notamment un autre épitope T CD8, un épitope T CD4 ou un épitope B de la NP et/ou de la GP du virus Ebola, ou bien un épitope d’un antigène d’un autre pathogène, notamment un virus de fièvre hémorragique. Parmi les épitopes B de la NP du virus Ebola on peut citer les peptides 173-187, 361 -375, 365-379, 381 -395, 417-431 , 425-439, 485-499 et 505-515 d’EBOV, lesdites positions étant indiquées en référence à la séquence SEQ ID NO : 1. Parmi les épitopes B de la GP du virus Ebola on peut citer les peptides 41 -55, 52- 66, 93-107, 112-126, 201-215, 217-231 , 221 -235, 225-239, 236-250, 301-305, 309-323, 317-331 , 321 -335, 325-339, 329-343, 333-347, 337-351 , 341-355, 345- 359, 381 -395, 385-399, 389-403, 393-407, 397-411 et 469-483 d’EBOV, lesdites positions étant indiquée en référence à la séquence SEQ ID NO : 49. [0040] According to one embodiment of the invention, said composition comprises at least one other epitope, in particular another CD8 T epitope, a CD4 T epitope or a B epitope of the NP and / or of the GP of the Ebola virus, or else an epitope of an antigen of another pathogen, in particular a hemorrhagic fever virus. Among the B epitopes of the Ebola virus NP, mention may be made of EBOV peptides 173-187, 361 -375, 365-379, 381 -395, 417-431, 425-439, 485-499 and 505-515, said positions being indicated with reference to the sequence SEQ ID NO: 1. Among the B epitopes of the Ebola virus GP, mention may be made of peptides 41 -55, 52- 66, 93-107, 112-126, 201-215, 217-231, 221 -235, 225-239, 236-250, 301-305, 309-323, 317-331, 321 -335, 325-339, 329-343, 333-347, 337-351, 341- 355, 345- 359, 381 -395, 385-399, 389-403, 393-407, 397-411 and 469-483 of EBOV, said positions being indicated with reference to the sequence SEQ ID NO: 49.
[0041] De préférence, la dite composition comprend au moins un épitope T CD4 de la NP ou de la GP du virus Ebola inclus dans une séquence choisie parmi les séquences SEQ ID NO : 74 à 96 et 129 à 138 de la NP et les séquences SEQ ID NO : 97 à 128 et 139 à 148 de la GP. Ladite composition comprend avantageusement au moins un épitope T CD8 selon l’invention dont la séquence n’est pas incluse dans la séquence comprenant au moins un épitope T CD4 telle que définie ci-dessus, c’est-à-dire que tous les résidus de l’épitope T CD8 ne sont pas présents dans la séquence comprenant au moins un épitope T CD4. Ledit épitope T CD8 dont la séquence n’est pas incluse dans la séquence comprenant au moins un épitope T CD4 telle que définie ci-dessus est avantageusement sélectionné dans le groupe constitué par les séquences SEQ ID NO : 12-14, 28-31 , 38-41 et 46-48 de la NP et les séquences SEQ ID NO : 54, 56, 63, 64, 66 et 70 de la GP. De préférence ladite composition comprend au moins un épitope T CD8 selon l’invention dont la séquence est disjointe (non-chevauchante) de celle comprenant au moins un épitope T CD4 ; cela signifie que la séquence de l’épitope T CD8 ne possède aucun résidu d’acide aminé en commun la séquence comprenant au moins un épitope T CD4 telle que définie ci-dessus. Ledit épitope T CD8 selon l’invention dont la séquence est disjointe (non-chevauchante) de celle comprenant au moins un épitope T CD4 telle que définie ci-dessus est avantageusement sélectionné dans le groupe constitué par les séquences SEQ ID NO : 40, 41 , 47 et 48 de la NP et les séquences SEQ ID NO : 54, 63, 64, 66 et 70 de la GP. Preferably, said composition comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of the GP. Said composition advantageously comprises at least one CD8 T epitope according to the invention, the sequence of which is not included in the sequence comprising at least one CD4 T epitope as defined above, that is to say that all the residues of the CD8 T epitope are not present in the sequence comprising at least one CD4 T epitope. Said CD8 T epitope, the sequence of which is not included in the sequence comprising at least one CD4 T epitope as defined above, is advantageously selected from the group consisting of the sequences SEQ ID NO: 12-14, 28-31, 38-41 and 46-48 of the NP and the sequences SEQ ID NO: 54, 56, 63, 64, 66 and 70 of the GP. Preferably, said composition comprises at least one CD8 T epitope according to the invention, the sequence of which is separate (non-overlapping) from that comprising at least one CD4 T epitope; this means that the sequence of the CD8 T epitope does not have any amino acid residue in common the sequence comprising at least one CD4 T epitope as defined above. Said CD8 T epitope according to the invention, the sequence of which is separate (non-overlapping) from that comprising at least one CD4 T epitope as defined above is advantageously selected from the group consisting of the sequences SEQ ID NO: 40, 41 , 47 and 48 of the NP and the sequences SEQ ID NO: 54, 63, 64, 66 and 70 of the GP.
[0042] Selon un mode de réalisation de l’invention, ladite combinaison d’épitopes T CD8, et éventuellement les autres épitopes tels que définis ci-dessus sont inclus dans un ou plusieurs : [0042] According to one embodiment of the invention, said combination of CD8 T epitopes, and optionally the other epitopes as defined above are included in one or more:
(a) peptides issus de la protéine NP de séquence SEQ ID NO : 1 ou GP de séquence SEQ ID NO : 49 ; (a) peptides derived from the NP protein of sequence SEQ ID NO: 1 or GP of sequence SEQ ID NO: 49;
(b) polypeptides multi-épitopiques ou protéines chimériques ; lesdits peptides en (a), polypeptides et protéines en (b) pouvant être éventuellement modifiés ; (b) multi-epitope polypeptides or chimeric proteins; said peptides in (a), polypeptides and proteins in (b) possibly being optionally modified;
(c) polynucléotides ou vecteurs recombinants codant le ou lesdits peptides, polypeptides et/ou protéines ; (c) recombinant polynucleotides or vectors encoding said peptide (s), polypeptides and / or proteins;
(d) un mélange dudit ou desdits peptide(s), polypeptide(s), protéine(s) de fusion, polynucléotide(s), vecteurs définis en (a) à (c). (d) a mixture of said peptide (s), polypeptide (s), fusion protein (s), polynucleotide (s), vectors defined in (a) to (c).
[0043] Dans un mode de réalisation avantageux de l’invention, la composition immunogène ou vaccinale comprend au moins un peptide issu de la NP ou GP ou un mélange de peptides issus de la NP et/ou GP ; ledit mélange comprenant avantageusement au plus 25 peptides ; de préférence 5 à 15 peptides. De préférence, ledit peptide ou mélange de peptides est apte à être présenté par au moins 8 (8, 9, 10, 11 , 12, 13 ou 14) de préférence au moins 12 (12, 13 ou 14) molécules HLA I prépondérantes différentes telles que définies ci-dessus, de manière préférée la totalité desdites molécules HLA I prépondérantes. [0043] In an advantageous embodiment of the invention, the immunogenic or vaccine composition comprises at least one peptide derived from NP or GP or a mixture of peptides derived from NP and / or GP; said mixture advantageously comprising at most 25 peptides; preferably 5 to 15 peptides. Preferably, said peptide or mixture of peptides is capable of being presented by at least 8 (8, 9, 10, 11, 12, 13 or 14), preferably at least 12 (12, 13 or 14) different predominant HLA I molecules as defined above, preferably all of said predominant HLA I molecules.
[0044] Dans ce mode de réalisation avantageux, chacun desdits peptides de la composition est de préférence un peptide d’au plus 100 acides aminés issu de la NP de séquence SEQ ID NO : 1 comprenant au moins deux à quatre séquences comprenant au moins un épitope T CD8 de la NP choisies parmi les séquences SEQ ID NO : 2 à 48 ou un peptide d’au plus 100 acides aminés issu de la GP de séquence SEQ ID NO : 49 comprenant au moins deux à quatre séquences comprenant au moins un épitope T CD8 de la GP choisies parmi les séquences SEQ ID NO : 50 à 73. De préférence, ledit peptide comprend 3 à 10 (3, 4, 5, 6, 7, 8, 9, 10) desdites séquences comprenant au moins un épitope T CD8 selon l’invention. De manière préférée, ladite composition comprend au moins un épitope T CD4 de la NP ou de la GP du virus Ebola inclus dans une séquence choisie parmi les séquences SEQ ID NO : 74 à 96 et 129 à 138 de la NP et les séquences SEQ ID NO : 97 à 128 et 139 à 148 de la GP, le ou lesdits épitopes T CD4 et T CD8 étant dans les mêmes peptides ou dans des peptides séparés et ladite composition comprenant avantageusement au moins un épitope T CD8 dont la séquence n’est pas incluse dans la séquence comprenant au moins un épitope T CD4 de la NP ou de la GP telle que définie ci-dessus, de préférence un épitope T CD8 dont la séquence n’est pas chevauchante avec la séquence comprenant au moins un épitope T CD4 de la NP ou de la GP telle que définie ci-dessus. In this advantageous embodiment, each of said peptides of the composition is preferably a peptide of at most 100 amino acids derived from the NP of sequence SEQ ID NO: 1 comprising at least two to four sequences comprising at least one CD8 T epitope of the NP chosen from the sequences SEQ ID NO: 2 to 48 or a peptide of at most 100 amino acids derived from the GP of sequence SEQ ID NO: 49 comprising at least two to four sequences comprising at least one CD8 T epitope of the GP chosen from the sequences SEQ ID NO: 50 to 73. Preferably, said peptide comprises 3 to 10 (3, 4, 5, 6, 7, 8, 9, 10) of said sequences comprising at least one CD8 T epitope according to the invention. Preferably, said composition comprises at least one CD4 T epitope of the NP or of the GP of the Ebola virus included in a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128 and 139 to 148 of GP, said T CD4 and T CD8 epitope (s) being in the same peptides or in separate peptides and said composition advantageously comprising at least one CD8 T epitope whose sequence is not included in the sequence comprising at least one CD4 T epitope of NP or GP as defined above, preferably a CD8 T epitope, the sequence of which does not overlap with the sequence comprising at least one CD4 T epitope of NP or GP as defined above.
[0045] Dans ce mode de réalisation avantageux, chacun desdits peptides de la composition consiste de préférence en une séquence de plus de 20 acides aminés à au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés) consécutifs de la séquence SEQ ID NO : 1 ou SEQ ID NO : 49, de manière préférée de 25 à 70 acides aminés consécutifs de la séquence SEQ ID NO : 1 ou SEQ ID NO : 49 ou une séquence de plus de 20 acides aminés à au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés), de manière préférée de 25 à 70 acides aminés possédant au moins 70%, d’identité, de préférence au moins 75%, 80%, 85% 90%, 95%, 98% ou 99% % d’identité avec la séquence SEQ ID NO : 1 ou SEQ ID NO : 49. In this advantageous embodiment, each of said peptides of the composition preferably consists of a sequence of more than 20 amino acids with at most 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 consecutive amino acids) of the sequence SEQ ID NO: 1 or SEQ ID NO: 49 , preferably from 25 to 70 consecutive amino acids of the sequence SEQ ID NO: 1 or SEQ ID NO: 49 or a sequence of more than 20 amino acids with at most 100 amino acids (21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids), preferably from 25 to 70 acids amines having at least 70% identity, preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99%% identity with the sequence SEQ ID NO: 1 or SEQ ID NO : 49.
[0046] Une composition préférée selon l’invention comprend au moins un peptide issu de ladite NP sélectionné dans le groupe constitué par : A preferred composition according to the invention comprises at least one peptide derived from said NP selected from the group consisting of:
a) les séquences SEQ ID NO : 149, 150, 151 et 152, a) the sequences SEQ ID NO: 149, 150, 151 and 152,
b) les séquences d’au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés) consécutifs de la séquence SEQ ID NO : 1 , de préférence de 25 à 70 acides aminés consécutifs de la séquence SEQ ID NO : 1 comprenant au moins l’une desdites séquences en a), et b) sequences of at most 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids) consecutive of the sequence SEQ ID NO: 1, preferably from 25 to 70 consecutive amino acids of the sequence SEQ ID NO: 1 comprising at least one of said sequences in a), and
c) les séquences d’au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés) consécutifs de la séquence SEQ ID NO : 1 , de préférence de 25 à 70 acides aminés possédant au moins 70% d’identité, de préférence au moins 75%, 80%, 85% 90%, 95%, 98% ou 99% % d’identité avec lesdites séquences en a). c) sequences of not more than 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 consecutive amino acids) of the sequence SEQ ID NO: 1, preferably 25 to 70 amino acids having at least 70% identity, preferably at least 75%, 80%, 85 % 90%, 95%, 98% or 99%% identity with said sequences in a).
[0047] De préférence, ladite composition comprend les peptides de séquence SEQ ID NO : 149, 150, 151 et 152 ou des peptides dérivés desdites séquences tels que définis en b) ou c). Les peptides de séquence SEQ ID NO : 149, 150 ,151 et 152 induisent des fréquences de répondeurs in vitro en T CD8+ spécifiques de la NP du virus Ebola, de respectivement 36%, 16%, 44% et 58%. Les peptides de séquence SEQ ID NO : 149, 150 et 151 induisent également des fréquences de répondeurs in vitro en T CD4+ spécifiques de la NP du virus Ebola, de respectivement 56,3%, 37,5% et 68,75% ([Tableau 12]). [0047] Preferably, said composition comprises peptides of sequence SEQ ID NO: 149, 150, 151 and 152 or peptides derived from said sequences as defined in b) or c). The peptides of sequence SEQ ID NO: 149, 150, 151 and 152 induce frequencies of in vitro CD8 + T responders specific for the NP of the Ebola virus, of 36%, 16%, 44% and 58%, respectively. The peptides of sequence SEQ ID NO: 149, 150 and 151 also induce frequencies of in vitro CD4 + T responders specific for the NP of the Ebola virus, of respectively 56.3%, 37.5% and 68.75% ([ Table 12]).
[0048] De préférence, la dite composition comprend également les peptides de séquence SEQ ID NO : 80 et 135 ; le peptide de séquence SEQ ID NO : 80 induit des fréquences de répondeurs in vitro en T CD8+ spécifiques et en T CD4+ de la NP du virus Ebola de respectivement 36% et 75%. Le peptide de séquence SEQ ID NO : 135 induit une fréquence de répondeurs in vitro en T CD4+ spécifiques de la NP du virus Ebola de 62,5% ([Tableau 12]). Preferably, said composition also comprises the peptides of sequence SEQ ID NO: 80 and 135; the peptide of sequence SEQ ID NO: 80 induces in vitro responder frequencies in specific CD8 + T and in CD4 + T of the NP of the Ebola virus of 36% and 75% respectively. The peptide of sequence SEQ ID NO: 135 induces a frequency of in vitro T-responders CD4 + specific for the NP of the Ebola virus of 62.5% ([Table 12]).
[0049] A titre d’exemple illustratif de cette composition préférée selon l’invention, on peut citer la composition comprenant les peptides NP de séquence SEQ ID NO : 80, 135, 149, 150, 151 et 152 ; cette composition est apte à induire une fréquence de répondeurs in vitro en T CD4+ spécifiques de 100 % et une fréquence maximale de répondeurs in vitro en T CD8+ spécifiques (92 % des individus du groupe de référence). Une telle composition permet d’obtenir une couverture vaccinale potentielle maximale de la population mondiale et en particulier des populations caucasiennes et africaines, en ce qui concerne l’immunité cellulaire contre le virus Ebola. [0050] Cet exemple qui est donné uniquement à titre illustratif n’est pas limitatif et d’autres compositions induisant des fréquences de réponse équivalentes peuvent être aisément obtenues avec d’autres combinaisons de peptides NP, déterminées à partir des profils de réponse en T CD8+ et en T CD4+ induits par respectivement les différents épitopes T CD8 [Tableau 2] et [Tableau 3] et les différents peptides comprenant des épitopes T CD4+ [Tableau 8], ainsi que les séquences des peptides NP présentés au [Tableau 12] By way of illustrative example of this preferred composition according to the invention, there may be mentioned the composition comprising the NP peptides of sequence SEQ ID NO: 80, 135, 149, 150, 151 and 152; this composition is capable of inducing a frequency of specific in vitro CD4 + T responders of 100% and a maximum frequency of specific in vitro CD8 + T responders (92% of the individuals of the reference group). Such a composition makes it possible to obtain a maximum potential vaccination coverage of the world population and in particular of the Caucasian and African populations, as regards cellular immunity against the Ebola virus. This example, which is given solely by way of illustration, is not limiting and other compositions inducing equivalent response frequencies can be easily obtained with other combinations of NP peptides, determined from the T response profiles CD8 + and CD4 + T respectively induced by the different CD8 T epitopes [Table 2] and [Table 3] and the different peptides comprising CD4 + T epitopes [Table 8], as well as the sequences of the NP peptides presented in [Table 12]
[0051] Une autre composition préférée selon l’invention comprend au moins un peptide issu de ladite GP sélectionné dans le groupe constitué par : Another preferred composition according to the invention comprises at least one peptide derived from said GP selected from the group consisting of:
a) les séquences SEQ ID NO : 153, 154, 155 et 156, a) the sequences SEQ ID NO: 153, 154, 155 and 156,
b) les séquences d’au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés) consécutifs de la séquence SEQ ID NO : 1 , de préférence de 25 à 70 acides aminés consécutifs de la séquence SEQ ID NO : 1 comprenant au moins l’une desdites séquences en a), et b) sequences of at most 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 consecutive amino acids) of the sequence SEQ ID NO: 1, preferably from 25 to 70 consecutive amino acids of the sequence SEQ ID NO: 1 comprising at least one of said sequences in a) , and
c) les séquences d’au plus 100 acides aminés (21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 ou 100 acides aminés) consécutifs de la séquence SEQ ID NO : 1 , de préférence de 25 à 70 acides aminés possédant au moins 70%, d’identité, de préférence au moins 75%, 80%, 85% 90%, 95%, 98% ou 99% % d’identité avec lesdites séquences en a). c) sequences of not more than 100 amino acids (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids) consecutive of the sequence SEQ ID NO: 1, preferably from 25 to 70 amino acids having at least 70%, identity, preferably at least 75%, 80%, 85% 90%, 95%, 98% or 99%% identity with said sequences in a).
[0052] De préférence, ladite composition comprend les peptides de séquence SEQ ID NO : 153, 154, 155 et 156 ou des peptides dérivés desdites séquences tels que définis en b) ou c). Les peptides de séquence SEQ ID NO : 153, 154, 155 et 156 induisent des fréquences de répondeurs in vitro en T CD8+ spécifiques de la GP du virus Ebola, de respectivement 12%, 24%, 24% et 20%. Les peptides de séquence SEQ ID NO : 153, 154, 155 et 156 induisent également des fréquences de répondeurs in vitro en T CD4+ spécifiques de la GP du virus Ebola, de respectivement 56,3%, 50%, 62,5% et 62,5% ([Tableau 13]). Preferably, said composition comprises peptides of sequence SEQ ID NO: 153, 154, 155 and 156 or peptides derived from said sequences as defined in b) or c). The peptides of sequence SEQ ID NO: 153, 154, 155 and 156 induce in vitro CD8 + T-responder frequencies specific for the GP of the Ebola virus, of 12%, 24%, 24% and 20%, respectively. The peptides of sequence SEQ ID NO: 153, 154, 155 and 156 also induce frequencies of in vitro T-responders CD4 + specific for the GP of the Ebola virus, respectively 56.3%, 50%, 62.5% and 62 , 5% ([Table 13]).
[0053] De préférence, la dite composition comprend également les peptides de séquence SEQ ID NO : 107 et 148 ; ces peptides induisent une fréquence de répondeurs in vitro en T CD4+ spécifiques de la GP du virus Ebola de respectivement 50% et 62,5% pour les peptides de séquence SEQ ID NO : 107 et 148. Preferably, said composition also comprises the peptides of sequence SEQ ID NO: 107 and 148; these peptides induce a frequency of in vitro T-responders CD4 + specific for the GP of the Ebola virus of 50% and 62.5% respectively for the peptides of sequence SEQ ID NO: 107 and 148.
[0054] A titre d’exemple illustratif de cette composition préférée selon l’invention, on peut citer la composition comprenant les peptides GP de séquence SEQ ID NO : 107, 148, 153, 154, 155 et 156 ; cette composition est apte à induire une fréquence de répondeurs in vitro en T CD4+ spécifiques de 100 % et une fréquence de répondeurs in vitro en T CD8+ spécifiques de 56%, proche de la fréquence maximale (60 % des individus du groupe de référence). Une telle composition permet d’obtenir une couverture vaccinale potentielle maximale de la population mondiale, et en particulier des populations caucasiennes et africaines, en ce qui concerne l’immunité cellulaire contre le virus Ebola. . As an illustrative example of this preferred composition according to the invention, mention may be made of the composition comprising the GP peptides of sequence SEQ ID NO: 107, 148, 153, 154, 155 and 156; this composition is capable of inducing a frequency of specific in vitro CD4 + T responders of 100% and a frequency of specific in vitro CD8 + T responders of 56%, close to the maximum frequency (60% of individuals in the reference group). Such a composition makes it possible to obtain a maximum potential vaccination coverage of the world population, and in particular of the Caucasian and African populations, with regard to cellular immunity against the Ebola virus. .
[0055] Cet exemple qui est donné uniquement à titre illustratif n’est pas limitatif et d’autres compositions induisant des fréquences équivalentes peuvent être aisément obtenues avec d’autres combinaisons de peptides GP, déterminées à partir des profils de réponse en T CD8+ et en T CD4+ induits par respectivement les différents épitopes T CD8 [Tableau 4] et les différents peptides comprenant des épitopes T CD4+ [Tableau 9], ainsi que les séquences des peptides GP présentés au [Tableau 13] This example, which is given solely by way of illustration, is not limiting and other compositions inducing equivalent frequencies can be easily obtained with other combinations of GP peptides, determined from the CD8 + T response profiles and in CD4 + T induced by respectively the different CD8 T epitopes [Table 4] and the different peptides comprising CD4 + T epitopes [Table 9], as well as the sequences of the GP peptides presented in [Table 13]
[0056] Selon un autre mode de réalisation de l’invention, la composition immunogène ou vaccinale comprend un polypeptide multiépitopique chimérique comprenant au moins une combinaison d’épitopes tels que définis ci-dessus. On définit ici un polypeptide chimérique comme une succession d’acides aminés qui n’est pas présente dans la nature. Un polypeptide multiépitopique chimérique selon l’invention comprend au moins deux à quatre épitopes T CD8 de la NP et/ou de la GP du virus Ebola tels que définis ci-dessus, les séquences desdits épitopes étant dans ledit polypeptide, adjacentes, liées par un élément de liaison ou séparées par une séquence comprenant un autre épitope tel que défini ci-dessus. L’autre épitope est notamment un autre épitope T CD8, un épitope T CD4 ou un épitope B de la NP et/ou de la GP du virus Ebola, ou bien un épitope d’un antigène d’un autre pathogène, notamment un virus de fièvre hémorrhagique. Le polypeptide multiépitopique peut également comprendre plusieurs copies d’une même séquence de peptide/épitope. Dans un mode de réalisation particulier, le polypeptide multiépitopique comprend moins de 100 acides aminés consécutifs de la NP de séquence SEQ ID NO : 1 et/ou de la GP de séquence SEQ ID NO : 49, de préférence moins de 50, de manière préférée moins de 30. Le polypeptide multiépitopique présente une longueur de 20 à 200 acides aminés, de préférence de 30 à 100 acides aminés, de manière préférée d’environ 50 acides aminés. According to another embodiment of the invention, the immunogenic or vaccine composition comprises a chimeric multi-epitope polypeptide comprising at least one combination of epitopes as defined above. A chimeric polypeptide is defined here as a succession of amino acids which is not present in nature. A chimeric multi-epitope polypeptide according to the invention comprises at least two to four CD8 T epitopes of the NP and / or of the GP of the Ebola virus as defined above, the sequences of said epitopes being in said polypeptide, adjacent, linked by a linking element or separated by a sequence comprising another epitope as defined above. The other epitope is in particular another CD8 T epitope, a CD4 T epitope or a B epitope of the NP and / or of the GP of the Ebola virus, or else an epitope of an antigen of another pathogen, in particular a virus. hemorrhagic fever. The multiepitope polypeptide can also comprise several copies of the same peptide / epitope sequence. In a particular embodiment, the Multiepitope polypeptide comprises less than 100 consecutive amino acids of the NP of sequence SEQ ID NO: 1 and / or of the GP of sequence SEQ ID NO: 49, preferably less than 50, more preferably less than 30. The multiepitope polypeptide has 20 to 200 amino acids in length, preferably 30 to 100 amino acids, more preferably about 50 amino acids.
[0057] Selon un autre mode de réalisation de l’invention, ledit peptide ou polypeptide tel que défini ci-dessus est un peptide ou un polypeptide modifié comprenant une modification au niveau de résidu(s) d’acide aminé, de la liaison peptidique ou de ses extrémités et ledit peptide ou polypeptide modifié conservant ses propriétés d’épitope T CD8 immunogène de la NP ou de la GP du virus Ebola telles que définies ci-dessus, c’est-à-dire qu’il est capable d’induire une réponse en lymphocytes T CD8 humains spécifiques chez des individus humains exprimant au moins une des molécules HLA I prépondérantes telles que définie ci- dessus. Cette ou ces modification(s), de préférence une ou des modification(s) chimique(s), qui sont introduites dans les peptides par les méthodes classiques connues de l’homme du métier, incluent de façon non-limitative l’une au moins des modifications chimiques suivantes : l’acétylation du résidu d’acide aminé N- terminal et/ou l’amidation du résidu d’acide aminé C-terminal (Maillère et al., Molecular Immunology, 1195, 32, 1377-1385), la substitution d’un acide aminé par un acide aminé non-protéinogénique (acide aminé D ou analogue d’acide aminé); l’addition de groupement chimique (lipide, oligo ou polysaccharide) au niveau d’une fonction réactive, notamment de la chaîne latérale R ; la modification de la liaison peptidique (-CO-NH-), notamment par une liaison du type rétro ou rétro- inverso (-NH-CO-) ou une liaison différente de la liaison peptidique ; la cyclisation ; la fusion de la séquence dudit peptide avec celle d’un peptide (épitope d’intérêt pour la vaccination); la fusion de la séquence dudit peptide avec celle d’une protéine, notamment une chaîne a d’une molécule HLA I ou le domaine extracellulaire de ladite chaîne, et le couplage à une molécule appropriée. Ces modifications sont destinées en particulier à augmenter la stabilité et plus particulièrement la résistance à la protéolyse, ainsi que la solubilité ou l’immunogénicité du peptide ou polypeptide selon l’invention. [0058] Selon un mode de réalisation avantageux dudit peptide ou polypeptide modifié, il comprend une ou plusieurs N6-acétyl-lysine(s), phosphosérine(s) et/ou phosphothréonine(s). According to another embodiment of the invention, said peptide or polypeptide as defined above is a peptide or a modified polypeptide comprising a modification at the level of amino acid residue (s), of the peptide bond or its ends and said modified peptide or polypeptide retaining its immunogenic CD8 T epitope properties of Ebola virus NP or GP as defined above, that is to say that it is capable of induce a response in specific human CD8 T lymphocytes in human individuals expressing at least one of the predominant HLA I molecules as defined above. This or these modification (s), preferably one or more chemical modification (s), which are introduced into the peptides by conventional methods known to those skilled in the art, include, without limitation, one of the following: less of the following chemical modifications: acetylation of the N-terminal amino acid residue and / or amidation of the C-terminal amino acid residue (Maillère et al., Molecular Immunology, 1195, 32, 1377-1385) , the substitution of an amino acid with a non-proteinogenic amino acid (D-amino acid or amino acid analog); the addition of a chemical group (lipid, oligo or polysaccharide) at the level of a reactive function, in particular of the side chain R; modification of the peptide bond (-CO-NH-), in particular by a bond of the retro or retro-inverso type (-NH-CO-) or a bond other than the peptide bond; cyclization; fusing the sequence of said peptide with that of a peptide (epitope of interest for vaccination); fusion of the sequence of said peptide with that of a protein, in particular an α chain of an HLA I molecule or the extracellular domain of said chain, and coupling to an appropriate molecule. These modifications are intended in particular to increase the stability and more particularly the resistance to proteolysis, as well as the solubility or the immunogenicity of the peptide or polypeptide according to the invention. According to an advantageous embodiment of said modified peptide or polypeptide, it comprises one or more N6-acetyl-lysine (s), phosphoserine (s) and / or phosphothreonine (s).
[0059] Selon un autre mode de réalisation avantageux, ledit peptide ou polypeptide modifié comprend l’acétylation de son résidu d’acide aminé N-terminal et/ou l’amidation de son résidu d’acide aminé C-terminal. [0059] According to another advantageous embodiment, said modified peptide or polypeptide comprises the acetylation of its N-terminal amino acid residue and / or the amidation of its C-terminal amino acid residue.
[0060] Selon un autre mode de réalisation avantageux, ledit peptide ou polypeptide modifié est un lipopeptide ou un lipopolypeptide. Ledit lipopeptide ou un lipopolypeptide peut être obtenu, notamment par addition d’un lipide sur une fonction a-aminée ou sur une fonction réactive de la chaîne latérale d’un acide aminé dudit peptide ou polypeptide; il peut comprendre une ou plusieurs chaînes dérivées d’acides gras en C4-20, éventuellement ramifiées ou insaturées (acide palmitique, acide oléique, acide linoléique, acide linolénique, acide 2-amino hexadécanoïque, pimélautide, trimétauxide) ou un dérivé d’un stéroïde. La partie lipidique préférée est notamment représentée par un groupe Na-acétyl-lysine NE(palmitoyl), également dénommé Ac-K(Pam). [0060] According to another advantageous embodiment, said modified peptide or polypeptide is a lipopeptide or a lipopolypeptide. Said lipopeptide or a lipopolypeptide can be obtained, in particular by adding a lipid to an α-amino function or to a reactive function of the side chain of an amino acid of said peptide or polypeptide; it may comprise one or more chains derived from C4-20 fatty acids, optionally branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-amino hexadecanoic acid, pimelautide, trimetalside) or a derivative of a steroid. The preferred lipid part is in particular represented by an N α -acetyl-lysine N E (palmitoyl) group, also called Ac-K (Pam).
[0061] Selon encore un autre mode de réalisation avantageux, la combinaison d’épitopes est incluse dans une protéine de fusion (protéine chimérique) comprenant ledit ou lesdits peptides fusionnés avec une protéine hétérologue (différente de la NP ou de la GP) ou un fragment polypeptidique hétérologue (fragment d’une protéine différente de NP ou de la GP dont la séquence n’est pas directement adjacente à la séquence dudit peptide dans la séquence de ladite NP ou GP). Ledit ou lesdits peptides peuvent être fusionnés avec l’extrémité NH2 ou COOH de ladite protéine ou dudit fragment polypeptidique hétérologue ou insérés dans la séquence de ladite protéine ou dudit fragment. According to yet another advantageous embodiment, the combination of epitopes is included in a fusion protein (chimeric protein) comprising said peptide or peptides fused with a heterologous protein (other than NP or GP) or a heterologous polypeptide fragment (fragment of a protein other than NP or GP, the sequence of which is not directly adjacent to the sequence of said peptide in the sequence of said NP or GP). Said peptide (s) may be fused with the NH2 or COOH end of said protein or said heterologous polypeptide fragment or inserted into the sequence of said protein or said fragment.
[0062] Selon une disposition avantageuse, ladite protéine chimérique est constituée par un ou plusieurs peptides ou polypeptides tels que défini ci-dessus, fusionnés avec l’une des chaînes d’une molécule HLA, de préférence la chaîne alpha d’une molécule HLA I, ou bien avec un fragment de celle-ci correspondant à une molécule HLA soluble, notamment un fragment correspondant au domaine extracellulaire précédé du peptide signal homologue ou d’un peptide signal hétérologue. Ledit peptide est avantageusement inséré entre le peptide signal et l’extrémité NH2 du domaine extracellulaire de la chaîne a, comme décrit pour la molécule HLA-A2 (Oved K et al. Cancer Immunol Immunother (2005) 54: 867- 879). According to an advantageous arrangement, said chimeric protein consists of one or more peptides or polypeptides as defined above, fused with one of the chains of an HLA molecule, preferably the alpha chain of an HLA molecule. I, or else with a fragment thereof corresponding to a soluble HLA molecule, in particular a fragment corresponding to the extracellular domain preceded by the homologous signal peptide or by a signal peptide heterologous. Said peptide is advantageously inserted between the signal peptide and the NH 2 end of the extracellular domain of the α chain, as described for the HLA-A2 molecule (Oved K et al. Cancer Immunol Immunother (2005) 54: 867- 879).
[0063] La présente invention a également pour objet une composition immunogène ou vaccinale comprenant au moins un polynucléotide codant pour au moins un peptide, polypeptide, et/ou ou protéine chimérique tels que définis ci- dessus. Ledit polynucléotide, est un ADN, un ARN ou un mélange d’ADN et d’ARN, recombinant ou synthétique. La séquence de l’ADN peut avantageusement être modifiée de façon à ce que l’usage des codons soit optimal chez l’hôte dans lequel elle est exprimée. A subject of the present invention is also an immunogenic or vaccine composition comprising at least one polynucleotide encoding at least one peptide, polypeptide, and / or or chimeric protein as defined above. Said polynucleotide is DNA, RNA or a mixture of DNA and RNA, recombinant or synthetic. The DNA sequence can advantageously be modified so that the use of codons is optimal in the host in which it is expressed.
[0064] Ces polynucléotides peuvent être insérés dans un vecteur d’expression, sous le contrôle transcriptionnel d’un promoteur approprié, pour permettre l’expression du ou desdits peptide(s) ou polypeptide(s) conforme à l'invention dans une cellule de l’hôte. These polynucleotides can be inserted into an expression vector, under the transcriptional control of an appropriate promoter, to allow the expression of said peptide (s) or polypeptide (s) in accordance with the invention in a cell from the host.
[0065] De nombreux vecteurs sont connus en eux-mêmes ; le choix d’un vecteur approprié dépend de l’utilisation envisagée pour ce vecteur (par exemple réplication de la séquence d’intérêt, expression de cette séquence, maintien de cette séquence sous forme extrachromosomique, ou bien intégration dans le matériel chromosomique de l’hôte), ainsi que de la nature de la cellule hôte. Par exemple, on peut utiliser des acides nucléiques nus (ADN ou ARN) ou des vecteurs viraux ou bactériens. Les vecteurs viraux sont notamment les adénovirus, les rétrovirus, les lentivirus et les AAV dans lesquels a été insérée préalablement la séquence d’intérêt ; on peut également associer ladite séquence (isolée ou insérée dans un vecteur plasmidique) avec une substance lui permettant de franchir la membrane des cellules hôtes, telle qu’un transporteur comme un nanotransporteur ou une préparation de liposomes, ou de polymères cationiques, ou bien l’introduire dans ladite cellule hôte en utilisant des méthodes physiques telles que l’électroporation ou la microinjection. En outre, on peut avantageusement combiner ces méthodes, par exemple en utilisant l’électroporation associée à des liposomes. [0066] De préférence, ledit vecteur est un vecteur d’expression comprenant tous les éléments nécessaires à l'expression du peptide, polypeptide, protéine de fusion tels que définis ci-dessus. Par exemple, ledit vecteur comprend une cassette d’expression incluant au moins un polynucléotide tel que défini ci-dessus, sous le contrôle de séquences régulatrices de la transcription et éventuellement de la traduction appropriées (promoteur, activateur, intron, codon d'initiation (ATG), codon stop, signal de polyadénylation, site d’épissage). Many vectors are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or else integration into the chromosomal material of the host), as well as the nature of the host cell. For example, naked nucleic acids (DNA or RNA) or viral or bacterial vectors can be used. The viral vectors are in particular adenoviruses, retroviruses, lentiviruses and AAVs in which the sequence of interest has been inserted beforehand; it is also possible to combine said sequence (isolated or inserted into a plasmid vector) with a substance allowing it to cross the membrane of host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or else the 'introducing into said host cell using physical methods such as electroporation or microinjection. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes. Preferably, said vector is an expression vector comprising all the elements necessary for the expression of the peptide, polypeptide, fusion protein as defined above. For example, said vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate transcriptional and possibly translation regulatory sequences (promoter, activator, intron, initiation codon ( ATG), stop codon, polyadenylation signal, splice site).
[0067] Selon un mode de réalisation avantageux de l’invention, ladite composition comprend au moins un polynucléotide codant ledit ou lesdits peptide(s), polypeptide(s), et/ou protéine(s) de fusion tels que définis ci-dessus, inséré dans un acide nucléique nu, de préférence un ADN nu. According to an advantageous embodiment of the invention, said composition comprises at least one polynucleotide encoding said peptide (s), polypeptide (s), and / or fusion protein (s) as defined above , inserted into naked nucleic acid, preferably naked DNA.
[0068] D’autres compositions conformes à l'invention peuvent également comprendre une cellule présentatrice d’antigène modifiée, comprenant au moins un peptide, polypeptide, protéine chimérique, polynucléotide et/ou un vecteur tels que définis ci-dessus, la cellule pouvant être modifiée de façon stable ou transitoire. La cellule est notamment une cellule présentatrice d’antigène naturelle telle qu’une cellule dendritique ou une cellule présentatrice de l’antigène artificielle, telle que des exosomes dérivés de cellules dendritiques ou des vésicules dérivées de cellules exprimant la NP ou la GP du virus Ebola ou des fragments immunogènes desdites protéines. La composition selon l’invention peut comprendre une cellule dendritique chargée avec au moins un peptide, polypeptide ou protéine chimérique selon l’invention, ou transformée avec au moins un polynucléotide ou un vecteur selon l’invention. Other compositions in accordance with the invention can also comprise a modified antigen presenting cell, comprising at least one peptide, polypeptide, chimeric protein, polynucleotide and / or a vector as defined above, the cell being able to be modified in a stable or transient manner. The cell is in particular a natural antigen presenting cell such as a dendritic cell or an artificial antigen presenting cell, such as exosomes derived from dendritic cells or vesicles derived from cells expressing NP or GP of the Ebola virus. or immunogenic fragments of said proteins. The composition according to the invention can comprise a dendritic cell loaded with at least one peptide, polypeptide or chimeric protein according to the invention, or transformed with at least one polynucleotide or a vector according to the invention.
[0069] La composition immunogène ou vaccinale selon l’invention comprend avantageusement un véhicule pharmaceutiquement acceptable, une substance porteuse et/ou un adjuvant. [0069] The immunogenic or vaccine composition according to the invention advantageously comprises a pharmaceutically acceptable vehicle, a carrier substance and / or an adjuvant.
[0070] Les véhicules pharmaceutiquement acceptables, les substances porteuses et les adjuvants sont ceux classiquement utilisés. Pharmaceutically acceptable vehicles, carrier substances and adjuvants are those conventionally used.
[0071] Les adjuvants sont avantageusement choisis parmi: les émulsions huileuses, les substances minérales, les extraits bactériens, les oligonucléotides contenant des CpG, la saponine, l’hydroxyde d’alumine, le monophosphoryl - lipide A et le squalène. The adjuvants are advantageously chosen from: oily emulsions, mineral substances, bacterial extracts, oligonucleotides containing CpGs, saponin, alumina hydroxide, monophosphoryl - lipid A and squalene.
[0072] Les substances porteuses sont avantageusement sélectionnées dans le groupe constitué par : les liposomes unilamellaires ou multilamellaires, les ISCOMS, les virosomes, les pseudoparticules virales, les micelles de saponine, les microsphères solides de nature saccharidique (poly(lactide-co-glycolide)) ou aurifère, et les nano-particules. The carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, solid microspheres of saccharide nature (poly (lactide-co-glycolide )) or gold, and nano-particles.
[0073] La composition conforme à l’invention peut comprendre en outre des adjuvants habituellement utilisés dans les vaccins, et permettant par exemple de favoriser l’administration du principe actif, de le stabiliser, d’augmenter son immunogénicité. The composition in accordance with the invention may further comprise adjuvants usually used in vaccines, and for example making it possible to promote the administration of the active principle, to stabilize it, to increase its immunogenicity.
[0074] Selon un mode de réalisation préféré de l’invention, la composition comprend en outre un antigène du virus Ebola, notamment la GP, sous forme d’une protéine recombinante, d’un polynucléotide ou d’un vecteur recombinant codant ledit antigène, ledit antigène d’Ebola étant éventuellement combiné à un adjuvant. La composition selon l’invention comprend avantageusement une combinaison d’épitopes T CD8 et T CD4 de la NP du virus Ebola et une protéine GP recombinante du virus Ebola, un polynucléotide, et/ou un vecteur codant ledit antigène, éventuellement combiné à un adjuvant. According to a preferred embodiment of the invention, the composition further comprises an antigen of the Ebola virus, in particular GP, in the form of a recombinant protein, of a polynucleotide or of a recombinant vector encoding said antigen. , said Ebola antigen optionally being combined with an adjuvant. The composition according to the invention advantageously comprises a combination of T CD8 and T CD4 epitopes of the NP of the Ebola virus and a recombinant GP protein of the Ebola virus, a polynucleotide, and / or a vector encoding said antigen, optionally combined with an adjuvant. .
[0075] La composition immunogène ou vaccinale comprend une dose efficace d’un ou plusieurs peptide(s), polypeptide(s), protéine(s), polynucléotide(s), et/ou vecteur(s), permettant d'obtenir un effet préventif sur une infection par un virus Ebola. Cette dose est déterminée et ajustée en fonction de facteurs tels que l’âge, le sexe et le poids du sujet. La composition est généralement administrée selon les protocoles usuels de vaccination, à des doses et pendant une durée suffisante pour induire une réponse cellulaire en lymphocytes T CD8+, et éventuellement T CD4+ dirigés contre la protéine NP et/ou GP du virus Ebola. L'administration peut être orale, parentérale ou locale, de préférence par injection, notamment sous- cutanée ou intramusculaire, La composition se présente sous une forme galénique adaptée à une administration choisie. [0076] La composition selon la présente invention est avantageusement utilisée comme vaccin pour la prévention des infections par le virus Ebola, notamment Ebola Zaïre, Soudan, Reston, Bundibuygo et Tai Forest ; de préférence Ebola Zaïre. The immunogenic or vaccine composition comprises an effective dose of one or more peptide (s), polypeptide (s), protein (s), polynucleotide (s), and / or vector (s), making it possible to obtain a preventive effect on infection with an Ebola virus. This dose is determined and adjusted according to factors such as the age, sex and weight of the subject. The composition is generally administered according to the usual vaccination protocols, at doses and for a period sufficient to induce a cellular response in CD8 + T lymphocytes, and optionally CD4 + T lymphocytes directed against the NP and / or GP protein of the Ebola virus. The administration can be oral, parenteral or local, preferably by injection, in particular subcutaneous or intramuscular. The composition is in a galenic form suitable for a chosen administration. The composition according to the present invention is advantageously used as a vaccine for the prevention of infections by the Ebola virus, in particular Ebola Zaire, Sudan, Reston, Bundibuygo and Tai Forest; preferably Ebola Zaire.
[0077] La présente invention a également pour objet une méthode de vaccination contre le virus Ebola, caractérisée en ce qu’elle comprend l’administration d’une composition vaccinale telle que définie ci-dessus, à un individu, par tout moyen approprié tel que défini ci-dessus. A subject of the present invention is also a method of vaccination against the Ebola virus, characterized in that it comprises the administration of a vaccine composition as defined above, to an individual, by any suitable means such as as defined above.
[0078] L’administration de la composition selon l’invention à un individu, induit une réponse une réponse cellulaire en lymphocytes T CD8, et éventuellement en lymphocytes T CD4, dirigés contre la protéine NP et/ou GP du virus Ebola. The administration of the composition according to the invention to an individual induces a cellular response in CD8 T lymphocytes, and optionally in CD4 T lymphocytes, directed against the NP and / or GP protein of the Ebola virus.
[0079] Selon un mode de réalisation, ladite composition immunogène ou vaccinale est utilisée en combinaison avec une autre composition vaccinale contre le virus Ebola, notamment une composition comprenant une protéine recombinante GP ou un polynucléotide ou vecteur codant ladite protéine. Lesdites compositions vaccinales sont utilisées simultanément, séparément ou de façon séquentielle. According to one embodiment, said immunogenic or vaccine composition is used in combination with another vaccine composition against the Ebola virus, in particular a composition comprising a recombinant GP protein or a polynucleotide or vector encoding said protein. Said vaccine compositions are used simultaneously, separately or sequentially.
[0080] La présente invention a également pour objet l’utilisation de la composition immunogène ou vaccinale selon la présente description pour la préparation d’un médicament (vaccin) destiné à la prévention des infections par le virus Ebola. [0080] A subject of the present invention is also the use of the immunogenic or vaccine composition according to the present description for the preparation of a medicament (vaccine) intended for the prevention of infections by the Ebola virus.
[0081] La présente invention a également pour objet l’utilisation in vitro de la composition telle que définie ci-dessus comme réactif pour le diagnostic d’une infection par le virus Ebola. A subject of the present invention is also the in vitro use of the composition as defined above as a reagent for the diagnosis of an infection by the Ebola virus.
[0082] La présente invention a également pour objet l’utilisation in vitro de la composition elle que définie ci-dessus comme réactif pour l’immunomonitorage de la réponse cellulaire contre le virus Ebola chez un individu ayant été exposé au virus Ebola ou ayant été vacciné contre le virus Ebola. A subject of the present invention is also the in vitro use of the composition as defined above as a reagent for the immunomonitoring of the cellular response against the Ebola virus in an individual who has been exposed to the Ebola virus or who has been vaccinated against Ebola virus.
[0083] La présente invention a également pour objet une méthode in vitro, d'immunomonitorage de la réponse cellulaire contre le virus Ebola chez un individu ayant été exposé au virus Ebola ou ayant été vacciné contre le virus Ebola, caractérisée en ce qu’elle comprend : - la mise en contact d'un échantillon biologique dudit individu comprenant des lymphocytes T CD8+ ou un mélange de lymphocytes T CD8+ et T CD4+, avec une composition selon l’invention telle que définie ci-dessus, et The present invention also relates to an in vitro method for immunomonitoring the cellular response against the Ebola virus in an individual who has been exposed to the Ebola virus or who has been vaccinated against the Ebola virus, characterized in that it includes: - bringing a biological sample of said individual comprising CD8 + T lymphocytes or a mixture of CD8 + T and CD4 + T lymphocytes into contact with a composition according to the invention as defined above, and
- la détection de lymphocytes T CD8+, et éventuellement de lymphocytes T CD4+, spécifiques du virus Ebola, par tout moyen approprié. the detection of CD8 + T lymphocytes, and optionally of CD4 + T lymphocytes, specific for the Ebola virus, by any appropriate means.
[0084] L’individu est de préférence un individu humain. L’échantillon biologique est notamment du sang total, des cellules mononuclées du sang périphériques (PBMC) isolés, des lymphocytes, des lymphocytes T CD8+ ou un mélange de lymphocytes T CD8+ et T CD4+ isolés, notamment à partir de PBMC. [0084] The individual is preferably a human individual. The biological sample is in particular whole blood, isolated peripheral blood mononuclear cells (PBMC), lymphocytes, CD8 + T lymphocytes or a mixture of isolated CD8 + and CD4 + T lymphocytes, in particular from PBMC.
[0085] La présente invention a également pour objet un réactif de diagnostic ou d’immunomonitorage comprenant la composition telle que définie ci-dessus. De préférence, ledit réactif comprend au moins un peptide, polypeptide ou protéine chimérique tels que définis ci-dessus, par exemple marqué et/ou complexé à une molécule HLA, notamment complexé à des molécules HLA marquées, par exemple biotinylées, sous la forme de complexes multimériques HLA/peptide tels que des tétramères de complexes HLA/peptide, marqués. Les dites molécules HLA marquées sont notamment des molécules HLA I marquées, un mélange de molécules HLA I marquées ou un mélange de molécules HLA I et HLA II marquées. De préférence, ledit réactif est inclus dans un coffret (kit). A subject of the present invention is also a diagnostic or immunomonitoring reagent comprising the composition as defined above. Preferably, said reagent comprises at least one peptide, polypeptide or chimeric protein as defined above, for example labeled and / or complexed with an HLA molecule, in particular complexed with labeled HLA molecules, for example biotinylated, in the form of HLA / peptide multimeric complexes such as tetramers of HLA / peptide complexes, labeled. Said labeled HLA molecules are in particular labeled HLA I molecules, a mixture of labeled HLA I molecules or a mixture of labeled HLA I and HLA II molecules. Preferably, said reagent is included in a box (kit).
[0086] La présente invention a ainsi, en outre, pour objet un procédé d’analyse de lymphocytes T CD8+ ou d’un mélange de lymphocytes T CD8+ et T CD4+, spécifiques du virus Ebola, caractérisé en ce qu’il comprend au moins les étapes suivantes : The present invention thus further relates to a method for analyzing CD8 + T lymphocytes or a mixture of CD8 + and CD4 + T lymphocytes, specific for the Ebola virus, characterized in that it comprises at least the following steps:
- la mise en contact, in vitro, d'un échantillon cellulaire comprenant des lymphocytes T CD8+ ou un mélange de lymphocytes T CD8+ et T CD4+ avec des complexes multimériques HLA /peptide, marqués, notamment par un fluorochrome, lesdits complexes étant formés par la liaison de molécules HLA solubles avec au moins un peptide, polypeptide ou protéine chimérique issu de la NP ou GP du virus Ebola Zaïre selon l’invention, et - bringing into contact, in vitro, a cell sample comprising CD8 + T lymphocytes or a mixture of CD8 + and CD4 + T lymphocytes with multimeric HLA / peptide complexes, labeled, in particular with a fluorochrome, said complexes being formed by the binding of soluble HLA molecules with at least one peptide, polypeptide or chimeric protein derived from the NP or GP of the Zaire Ebola virus according to the invention, and
- l’analyse des cellules liées auxdits complexes HLA l/peptide, notamment en cytométrie de flux. [0087] Selon un mode de mise en oeuvre avantageux dudit procédé, l’analyse des cellules (lymphocytes T CD8+ ou TCD8+ et T CD4+) comprend le tri desdites cellules. the analysis of cells linked to said HLA 1 / peptide complexes, in particular by flow cytometry. According to an advantageous embodiment of said method, the analysis of the cells (CD8 + or TCD8 + T and CD4 + T lymphocytes) comprises sorting said cells.
[0088] La présente invention a également pour objet un peptide constitué d’un épitope T CD8 immunogène de la NP ou GP du virus Ebola de séquence SEQ ID NO : 3, 4, 6, 9-13, 15-16, 18, 20, 22-28, 30-34, 37, 39-42, 44, 46, 47, 50 à 73 et 150 à 156 tel que définis ci-dessus. A subject of the present invention is also a peptide consisting of an immunogenic CD8 T epitope of the NP or GP of the Ebola virus of sequence SEQ ID NO: 3, 4, 6, 9-13, 15-16, 18, 20, 22-28, 30-34, 37, 39-42, 44, 46, 47, 50 to 73 and 150 to 156 as defined above.
[0089] Les peptides et leurs dérivés tels que définis ci-dessus, sont préparés par les techniques classiques connues de l'homme du métier. Par exemple, les peptides et leurs dérivés peuvent être synthétisés en phase solide, selon la technique Fmoc, originellement décrite par Merrifield et al. (J. Am. Chem. Soc., 1964, 85: 2149-) ou en phase liquide, et purifiés par chromatographie liquide haute performance en phase inverse. Les lipopeptides peuvent être notamment préparés selon le procédé décrit dans les Demandes Internationales WO 99/40113 ou WO 99/51630. Les peptides et t leurs dérivés tels que définis ci- dessus peuvent également être produits à partir des ADNc correspondants, obtenus par tout moyen connu de l’homme du métier ; l’ADNc est cloné dans un vecteur d’expression eucaryote ou procaryote et la protéine ou le fragment produits dans les cellules modifiées par le vecteur recombinant sont purifiés par tout moyen approprié, notamment par chromatographie d’affinité. The peptides and their derivatives as defined above are prepared by standard techniques known to those skilled in the art. For example, the peptides and their derivatives can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85: 2149-) or in liquid phase, and purified by reverse phase high performance liquid chromatography. The lipopeptides can in particular be prepared according to the process described in International Applications WO 99/40113 or WO 99/51630. The peptides and their derivatives as defined above can also be produced from the corresponding cDNAs, obtained by any means known to those skilled in the art; the cDNA is cloned into a eukaryotic or prokaryotic expression vector and the protein or the fragment produced in the cells modified by the recombinant vector are purified by any suitable means, in particular by affinity chromatography.
[0090] Les polynucléotides selon l'invention sont obtenus par les méthodes classiques, connues en elles-mêmes. Par exemple, ils peuvent être obtenus par amplification d'une séquence nucléique par PCR ou RT-PCR, par criblage de banques d'ADN génomique par hybridation avec une sonde homologue, ou bien par synthèse chimique totale ou partielle. Les vecteurs recombinants sont construits et introduits dans des cellules hôtes par les méthodes classiques d'ADN recombinant et de génie génétique, qui sont connues en elles-mêmes. The polynucleotides according to the invention are obtained by conventional methods, known in themselves. For example, they can be obtained by amplification of a nucleic acid sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or else by total or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.
Brève description des dessins Brief description of the drawings
[0091] La présente invention sera mieux comprise à l’aide du complément de description qui va suivre, qui se réfère à des exemples non-limitatifs illustrant l’identification et la caractérisation des épitopes T CD8 et T CD4 immunogènes issus de la nucléoprotéine (NP) et de la glycoprotéine (GP) du virus Ebola Zaïre selon la présente invention, ainsi qu’aux figures annexées, sur lesquels : The present invention will be better understood with the aid of the additional description which follows, which refers to non-limiting examples illustrating the identification and characterization of the immunogenic CD8 T and CD4 T epitopes. derived from the nucleoprotein (NP) and the glycoprotein (GP) of the Zaire Ebola virus according to the present invention, as well as from the appended figures, in which:
Fig.1 Fig. 1
[0092] [Fig. 1 ] montre les fréquences alléliques (%) des molécules HLA I sélectionnées dans différentes populations Caucasiennes (France) et Africaines (Cameroun, Soudan, Afrique du Sud) et dans le groupe de référence testé (panel). [0092] [Fig. 1] shows the allele frequencies (%) of the HLA I molecules selected in different Caucasian (France) and African (Cameroon, Sudan, South Africa) populations and in the reference group tested (panel).
EXEMPLES EXAMPLES
EXEMPLE 1 : Identification d’épitopes T CD8+ du virus Ebola Matériels et méthodes EXAMPLE 1: Identification of CD8 + T epitopes of the Ebola virus Materials and methods
1. Design et production des peptides 1. Design and production of peptides
[0093] Les séquences de la NP (UniProt - AAD14590.1 ; SEQ ID NO : 1 ) et de la GP (UniProt - AKG65268.1 ; SEQ ID NO : 49) du virus Ebola proviennent du Centre américain pour les informations biotechnologiques (NCBI). La souche Zaïre de 1976 a été utilisée (ZEBOV). Les peptides ont été sélectionnés à l’aide de l’outil de prédiction de liaison au CMH-I, NetMHC v4.0. Pour l’allèle HLA-A*02, les peptides présentant un rang inférieur ou égal à 1 % ont été sélectionnés. Pour les autres allèles, seuls les dix meilleurs peptides (NP et GP confondus) ont été sélectionnés. Les peptides de 9-mers et 10-mers ont été synthétisés par Pepscan (Pays-Bas), avec une pureté minimale de 80%. The sequences of the NP (UniProt - AAD14590.1; SEQ ID NO: 1) and of the GP (UniProt - AKG65268.1; SEQ ID NO: 49) of the Ebola virus come from the American Center for Biotechnological Information ( NCBI). The Zaire strain from 1976 was used (ZEBOV). Peptides were selected using the MHC-I binding prediction tool, NetMHC v4.0. For the HLA-A * 02 allele, peptides with a rank less than or equal to 1% were selected. For the other alleles, only the ten best peptides (NP and GP combined) were selected. The 9-mer and 10-mer peptides were synthesized by Pepscan (Netherlands), with a minimum purity of 80%.
2. Préparation des cellules 2. Preparation of cells
[0094] Les prélèvements sanguins (Couche leuco plaquettaire) proviennent de l’Établissement Français du Sang (Centre de Rungis). Les PBMC ont été isolés par gradient de ficoll. Les cellules dendritiques (DC) immatures ont été obtenues à partir des PBMC par différenciation des cellules adhérentes après 5 jours de culture en milieu AIM-V contenant 1000 U/ml d’IL-4 et 1000 U/ml de GM-CSF (R&D Systems). Les DC matures ont été obtenues à partir des DC immatures après avoir été cultivées pendant deux jours en présence de LPS. Les lymphocytes T CD8+ ont été purifiés à partir des PBMC à l’aide de microbilles magnétiques ciblant toutes les populations non CD3+/CD8+ (Miltenyi Biotec). Le génotypage des donneurs a été réalisé par NGS par la société DKMS Life Science (Dreseden, Allemagne). The blood samples (buffy coat) come from the French Blood Establishment (Rungis Center). PBMCs were isolated by ficoll gradient. Immature dendritic cells (DC) were obtained from PBMCs by differentiation of adherent cells after 5 days of culture in AIM-V medium containing 1000 U / ml of IL-4 and 1000 U / ml of GM-CSF (R&D Systems). Mature DCs were obtained from immature DCs after being cultured for two days in the presence of LPS. The CD8 + T cells were purified from PBMCs using magnetic microbeads targeting all non-CD3 + / CD8 + populations (Miltenyi Biotec). The genotyping of the donors was carried out by NGS by the company DKMS Life Science (Dreseden, Germany).
3. Obtention de lignées de lymphocytes T CD8+ spécifiques des peptides 3. Obtaining peptide-specific CD8 + T lymphocyte lines
[0095] Les lymphocytes T CD8+ (1 à 3x105) ont été mis en culture en plaque 96 puits à fond rond avec des DC matures autologues préalablement chargées avec des pools de peptides (10 mg/ml). La culture est faite dans du milieu IMDM supplémenté par du Sérum AB (milieu IMDM complet) contenant 30 ng/mL d’IL-21 (Bio-techne). 20 lignées lymphocytaires ont été ensemencées pour chaque pool de peptides. La culture est stimulée au bout d’une semaine par ajout de 10000 à 30000 DC chargées avec les pools de peptides, 5 ng/mL IL-15 et 5 ng/mL IL-7 (Bio-techne). Entre 5 et 7 jours après la restimulation, un test de spécificité est réalisé par ELISpot. Chaque puits de culture constitue une lignée de lymphocytes T CD8+. The CD8 + T lymphocytes (1 to 3 × 10 5 ) were cultured in a 96-well round-bottom plate with mature autologous DCs previously loaded with pools of peptides (10 mg / ml). The culture is carried out in IMDM medium supplemented with Serum AB (complete IMDM medium) containing 30 ng / ml of IL-21 (Bio-techne). 20 lymphocyte lines were seeded for each peptide pool. The culture is stimulated after one week by adding 10,000 to 30,000 DC loaded with the peptide pools, 5 ng / ml IL-15 and 5 ng / ml IL-7 (Bio-techne). Between 5 and 7 days after restimulation, a specificity test is performed by ELISpot. Each culture well constitutes a line of CD8 + T lymphocytes.
4. Evaluation de la spécificité de lymphocytes T CD8 cultivés avec les peptides par ELISpot 4. Evaluation of the specificity of CD8 T lymphocytes cultured with the peptides by ELISpot
[0096] L’anticorps anti-IFN-g humain 1 -D1 K est adsorbé à 2,5mg/ml en PBS 1X (Mabtech) sur des plaques 96 puits Multiscreen HA (Millipore) par une incubation d’une nuit à 4°C. Les plaques sont ensuite saturées par une incubation de 2 heures à 37°C avec du milieu l’IMDM complet, Les lymphocytes T CD8+ sont incubés pendant 16 heures à 37°C dans ces plaques après avoir été lavés dans du milieu AIM-V/IL-7 (0,5ng/ml d’IL-7). Des PBMC (50 000 cellules/puits) incubés en présence de 10mg/ml de peptides sont utilisées comme cellules présentatrices. Après l’incubation, les plaques sont lavées avec de l’eau distillée, du PBS /Tween 0,05% et du PBS. 10OmI/puits d’Ac anti-IFN-g humain 7-B6-1 biotinylé (Mabtech) à 0,25mg/ml en PBS 1X/BSA 1 % est ajouté dans les plaques qui sont incubées 1 h30 à 37°C. Après plusieurs lavages en PBS ou PBS/tween 0,05%, la sécrétion d’IFN- Y est mise en évidence par l’ajout de l’Extravidine-Phosphatase Alcaline (Sigma) et de NBT/BCIP. Après 5 à 10 minutes d’incubation, la réaction est stoppée par lavage à l’eau courante. Après séchage, les spots sont comptés sur un lecteur ELISpot (AID). Une lignée de lymphocytes T CD8+ est considérée comme spécifiques de l’antigène si le nombre de spots dans les puits contenant l’antigène est au moins deux fois plus élevé que le puits ne contenant pas l’antigène, la différence entre les 2 types de puits étant au moins supérieure à 25. Résultats The anti-human IFN-g 1 -D1 K antibody is adsorbed at 2.5 mg / ml in 1X PBS (Mabtech) on 96-well Multiscreen HA plates (Millipore) by an overnight incubation at 4 ° vs. The plates are then saturated by a 2 hour incubation at 37 ° C with complete IMDM medium, CD8 + T lymphocytes are incubated for 16 hours at 37 ° C in these plates after having been washed in AIM-V medium / IL-7 (0.5ng / ml IL-7). PBMCs (50,000 cells / well) incubated in the presence of 10 mg / ml of peptides are used as presenting cells. After the incubation, the plates are washed with distilled water, PBS / 0.05% Tween and PBS. 10OmI / well of biotinylated anti-human IFN-g 7-B6-1 Ab (Mabtech) at 0.25 mg / ml in 1 × PBS / 1% BSA is added to the plates which are incubated for 1 h 30 min at 37 ° C. After several washes in PBS or PBS / 0.05% tween, the secretion of IFN-Y is demonstrated by the addition of Alkaline Extravidin-Phosphatase (Sigma) and of NBT / BCIP. After 5 to 10 minutes of incubation, the reaction is stopped by washing with running water. After drying, the spots are counted on a reader ELISpot (AID). A CD8 + T lymphocyte line is considered to be specific for the antigen if the number of spots in the wells containing the antigen is at least twice as high as the well not containing the antigen, the difference between the 2 types of wells being at least greater than 25. Results
1. Sélection des peptides par prédiction in silico 1. Selection of peptides by in silico prediction
[0097] Les peptides issus de la NP et de la GP d’Ebola Zaïre ont été sélectionnés à l’aide de l’outil de prédiction de liaison au CMH-I, NetMHC v4.0. Les allèles HLA- A et HLA-B présents chez au moins 5% des individus dans différentes populations ont été recherchés. Les allèles HLA-C n’ont pas été retenus en raison de la faible expression de ces derniers en comparaison des allèles HLA-A et HLA-B (Neefjes et Ploegh, Eur. J. Immunol., 1988, 18, 801-810). Les allèles HLA I sélectionnés sont : HLA-A*01 :01 , HLA-A*02:01 , HLA-A*03:01 , HLA-A*11 :01 , HLA-A*23 :01 , HLA-A*24:02, HLA-A*29:02, HLA-B*07:02, HLA-B*08:01 , HLA-B*15:01 , HLA- B*15:02, HLA-B*15:03, HLA-B*18:01 , HLA-B*35:01 , HLA-B*35:02, HLA-B*35:03,Peptides from Ebola Zaire NP and GP were selected using the MHC-I binding prediction tool, NetMHC v4.0. The HLA-A and HLA-B alleles present in at least 5% of individuals in different populations have been sought. The HLA-C alleles were not retained because of the low expression of the latter compared to the HLA-A and HLA-B alleles (Neefjes and Ploegh, Eur. J. Immunol., 1988, 18, 801-810 ). The selected HLA I alleles are: HLA-A * 01: 01, HLA-A * 02: 01, HLA-A * 03: 01, HLA-A * 11: 01, HLA-A * 23: 01, HLA-A * 24:02, HLA-A * 29: 02, HLA-B * 07: 02, HLA-B * 08: 01, HLA-B * 15: 01, HLA- B * 15: 02, HLA-B * 15 : 03, HLA-B * 18: 01, HLA-B * 35: 01, HLA-B * 35: 02, HLA-B * 35: 03,
HLA-B*40:01 , HLA-B*44:02, HLA-B*44:03, HLA-B*51 :01 ([Tableau 1 ]). HLA-B * 40:01, HLA-B * 44:02, HLA-B * 44:03, HLA-B * 51:01 ([Table 1]).
[0098] [Tableau 1] [0098] [Table 1]
Figure imgf000038_0001
[0099] 92 peptides pour la NP et 46 peptides pour la GP ont été sélectionnés par des méthodes de prédiction in silico pour être testés in vitro. La NP semble plus immunogène que la GP, le nombre de peptides candidats étant deux fois plus élevé, ce qui est concordant avec les données publiées par Mc Elroy et collaborateurs (McElroy et al., 2015, précité).
Figure imgf000038_0001
92 peptides for NP and 46 peptides for GP were selected by in silico prediction methods to be tested in vitro. NP appears to be more immunogenic than GP, the number of candidate peptides being twice as high, which is consistent with the data published by Mc Elroy et al. (McElroy et al., 2015, supra).
2. Obtention de lignées de lymphocytes T CD8+ spécifiques de la NP et de la GP de ZEBOV 2. Obtaining CD8 + T lymphocyte lines specific for NP and GP from ZEBOV
[0100] 25 donneurs sains comportant des molécules HLA variées et représentatives des populations caucasiennes et africaines ont été sélectionnés [Fig. 1] Il s’agit des allèles HLA-A*01 :01 , HLA-A*02:01 , HLA-A*03:01 , HLA- A*11 :01 , HLA-A*24:02, HLA-A*29:02, HLA-B*07:02, HLA-B*08:01 , HLA-B*15:01 , HLA-B*18:01 , HLA-B*35:01 , HLA-B*35:03, HLA-B*40:01 , HLA-B*44:02, HLA- B*44:03 et HLA-B*51 :01. [0100] 25 healthy donors comprising various HLA molecules representative of Caucasian and African populations were selected [Fig. 1] These are the alleles HLA-A * 01: 01, HLA-A * 02:01, HLA-A * 03:01, HLA- A * 11: 01, HLA-A * 24:02, HLA- A * 29:02, HLA-B * 07:02, HLA-B * 08:01, HLA-B * 15:01, HLA-B * 18: 01, HLA-B * 35: 01, HLA-B * 35:03, HLA-B * 40:01, HLA-B * 44: 02, HLA- B * 44: 03 and HLA-B * 51:01.
[0101] Des lignées de lymphocytes T CD8+ ont été obtenues par co-culture des lymphocytes T CD8+ avec des cellules dendritiques autologues préalablement chargées avec des pools de peptides. Après amplification des lymphocytes T, chaque lignée a été testée par ELISpot pour sa capacité à reconnaître des pools de peptides puis dans un second test, les peptides individuels contenus dans le pool reconnu par les lignées. CD8 + T lymphocyte lines were obtained by coculture of CD8 + T lymphocytes with autologous dendritic cells previously loaded with pools of peptides. After amplification of the T lymphocytes, each line was tested by ELISpot for its ability to recognize pools of peptides and then in a second test, the individual peptides contained in the pool recognized by the lines.
[0102] Le [Tableau 2] et le [Tableau 3] présentent les réponses individuelles en lymphocytes T CD8+ spécifiques contre les différents peptides de la NP et de la GP d’Ebola, respectivement. Les peptides (épitopes T CD8) reconnus pas les lymphocytes T CD8+ spécifiques de la NP et de la GP d’Ebola sont indiqués. Le typage HLA (Restriction) pour lequel le donneur a été testé, le nombre de donneurs testés, le nombre de donneurs positifs et le pourcentage de donneur répondeur par allèle sont également indiqués. [0103] [Tableau 2] [0102] [Table 2] and [Table 3] show the individual responses in specific CD8 + T lymphocytes against the different peptides of Ebola NP and GP, respectively. Peptides (CD8 T epitopes) recognized by CD8 + T lymphocytes specific for Ebola NP and GP are indicated. The HLA (Restriction) typing for which the donor has been tested, the number of donors tested, the number of positive donors and the percentage of responding donor per allele are also indicated. [0103] [Table 2]
Figure imgf000040_0001
[0104] [Tableau 3]
Figure imgf000040_0001
[0104] [Table 3]
Figure imgf000041_0001
[0105] [Tableau 4]
Figure imgf000041_0001
[0105] [Table 4]
Figure imgf000042_0001
[0106] Les tests d’amplification des lymphocytes T CD8+ sont effectués à partir d’un panel de donneurs sains possédant des molécules HLA de classe I variées. Les cellules T spécifiques étant présentes dans le sang avec une fréquence faible, les lymphocytes T sont amplifiés par des cycles de stimulation antigénique. La spécificité des cellules pour les peptides d’intérêt est évaluée par ELISpot. Ces tests permettent d’évaluer la réponse des lymphocytes vis-à-vis des peptides et d’ainsi sélectionner la combinaison de peptides ayant une fréquence de répondeurs élevée.
Figure imgf000042_0001
[0106] The CD8 + T lymphocyte amplification tests are carried out using a panel of healthy donors having various HLA class I molecules. Since specific T cells are present in the blood at a low frequency, T lymphocytes are amplified by cycles of antigen stimulation. The specificity of the cells for the peptides of interest is evaluated by ELISpot. These tests make it possible to evaluate the response of the lymphocytes to the peptides and thus to select the combination of peptides having a high responder frequency.
[0107] Un total de 54 et 28 épitopes ont été mis en évidence pour la N P et la GP respectivement, ce qui correspond à environ 60% des peptides testés pour chacune des protéines. Sur les 25 donneurs testés, des réponses lymphocytaires T CD8+ contre les peptides issus de la NP et de la GP ont été obtenues pour 23 et 15 donneurs, respectivement. Par ailleurs, il est à noter qu’aucun épitope restreint à l’allèle HLA-A*23 n’a été obtenu, que ce soit pour les peptides issus de la NP ou de la GP. Par ailleurs, il n’a pas été possible de mettre en évidence d’épitopes issu de la GP restreint à l’allèle HLA-B*08. [0107] A total of 54 and 28 epitopes were demonstrated for N P and GP respectively, which corresponds to approximately 60% of the peptides tested for each of the proteins. Of the 25 donors tested, CD8 + T lymphocyte responses against peptides from NP and GP were obtained for 23 and 15 donors, respectively. Furthermore, it should be noted that no epitope restricted to the HLA-A * 23 allele was obtained, either for the peptides derived from NP or GP. Furthermore, it was not possible to demonstrate epitopes from GP restricted to the HLA-B * 08 allele.
5. Conservations des épitopes 5. Preservation of epitopes
[0108] Comme, les peptides identifiés et étudiés sont issus de la souche Zaïre du virus Ebola, la conservation des séquences de ces peptides entre les différentes souches du virus Ebola a été étudiée. Les résultats sont représentés en pourcentage d’identité de séquence par rapport à la souche Zaïre. Le pourcentage d’identité des peptides entre les différentes souches est plus élevé pour les peptides de la NP que de la GP [Tableau 5] et [Tableau 6] Ces peptides, en particulier les peptides NP, devraient ainsi pouvoir être utilisés dans une composition vaccinale pour l’ensemble des souches du virus Ebola. [0109] [Tableau 5] Since the peptides identified and studied are derived from the Zaire strain of the Ebola virus, the conservation of the sequences of these peptides between the different strains of the Ebola virus has been studied. The results are represented as a percentage of sequence identity relative to the Zaire strain. The percentage identity of the peptides between the different strains is higher for the NP peptides than for the GP [Table 5] and [Table 6] These peptides, in particular the NP peptides, should thus be able to be used in a composition. vaccine for all strains of the Ebola virus. [0109] [Table 5]
Figure imgf000044_0001
[Tableau 6]
Figure imgf000044_0001
[Table 6]
Figure imgf000045_0001
Figure imgf000045_0001
EXEMPLE 2 : Identification d’épitopes T CD4+ du virus Ebola Matériels et méthodes EXAMPLE 2: Identification of CD4 + T epitopes of the Ebola virus Materials and methods
1. Design et production des peptides 1. Design and production of peptides
[0110] Les séquences de la NP (UniProt AAD14590.1 ; SEQ ID NO : 1 ) et de la GP (UniProt AKG65268.1 ; SEQ ID NO : 49) du virus Ebola proviennent du Centre américain pour les informations biotechnologiques (NCBI). La souche Zaïre de 1976 a été utilisée (ZEBOV). Les peptides ont été sélectionnés à l’aide de l’outil de prédiction de liaison au CMH-II, issu de la base de données IEDB (http://www.iedb.org/). Les algorithmes NetMHCIIpan et Sturniolo ont été utilisés pour prédire la capacité de liaison d’un peptide donné aux molécules HLA de classe II sélectionnées. Des peptides de 20-mers contenant les cœurs proches ont ensuite été définis, de manière à ce que les cœurs ne soient pas en position 1 ou en position 20 du peptide. Les peptides de 20-mers ont été synthétisés par Pepscan (Pays-Bas), avec une pureté minimale de 80%. [0110] The sequences of the NP (UniProt AAD14590.1; SEQ ID NO: 1) and of the GP (UniProt AKG65268.1; SEQ ID NO: 49) of the Ebola virus come from the American Center for Biotechnological Information (NCBI) . The Zaire strain from 1976 was used (ZEBOV). Peptides were selected using the MHC-II Binding Prediction Tool from the IEDB database (http://www.iedb.org/). The NetMHCIIpan and Sturniolo algorithms were used to predict the binding capacity of a given peptide to selected HLA class II molecules. 20-mer peptides containing the nearby cores were then defined, so that the cores were not in position 1 or in position 20 of the peptide. The 20-mer peptides were synthesized by Pepscan (Netherlands), with a minimum purity of 80%.
2. Préparation des cellules 2. Preparation of cells
[0111] Les prélèvements sanguins (Couche leuco plaquettaire) proviennent de l’Établissement Français du Sang (Centre de Rungis). Les cellules mononuclées de sang périphérique (PBMC) ont été isolées par gradient de ficoll. Les cellules dendritiques (DC) immatures ont été obtenues à partir des PBMC par différenciation des cellules adhérentes après 5 jours de culture en milieu AIM-V contenant 1000 U/ml d’IL-4 et 1000 U/ml de GM-CSF (R&D Systems). Les DC matures ont été obtenues à partir des DC immatures après avoir été cultivées pendant deux jours en présence de LPS. Les lymphocytes T CD4+ ont été purifiés à partir des PBMC à l’aide de microbilles magnétiques couplées à des anticorps anti-CD4 (Miltenyi Biotec). Le génotypage des donneurs a été réalisé par NGS par la société DKMS Life Science (Dresden, Allemagne). 3. Obtention de lignées de lymphocytes T CD4+ spécifiques des peptides [0111] The blood samples (buffy coat) come from the French Blood Establishment (Rungis Center). Peripheral blood mononuclear cells (PBMC) were isolated by ficoll gradient. Immature dendritic cells (DC) were obtained from PBMCs by differentiation of adherent cells after 5 days of culture in AIM-V medium containing 1000 U / ml of IL-4 and 1000 U / ml of GM-CSF (R&D Systems). Mature DCs were obtained from immature DCs after being cultured for two days in the presence of LPS. CD4 + T lymphocytes were purified from PBMCs using magnetic microbeads coupled to anti-CD4 antibodies (Miltenyi Biotec). The genotyping of the donors was carried out by NGS by the company DKMS Life Science (Dresden, Germany). 3. Obtaining peptide-specific CD4 + T lymphocyte lines
[0112] Les lymphocytes T CD4+ (1 à 3x105) ont été mis en culture en plaque 96 puits à fond rond avec des DC matures autologues préalablement chargées avec des pools de peptides (10 mg/ml). La culture est faite dans du milieu IMDM supplémenté par du Sérum AB (milieu IMDM complet) contenant 1000U/ml d’IL-6 (R&D Systems) et 10ng/ml d’IL-12 (R&D Systems). 25 lignées lymphocytaires ont été ensemencées pour chaque pool de peptides. La culture est stimulée au bout d’une semaine par ajout de 10000 à 30000 DC chargées avec les pools de peptides, de 20U/ml d’IL-2 (R&D Systems) et de 10ng/ml d’IL-7 (R&D Systems). Une autre stimulation est faite après 14 et 21 jours de culture. Entre 5 et 7 jours après la dernière stimulation, un test de spécificité est réalisé par ELISpot. Chaque puits de culture constitue une lignée de lymphocytes T CD4+. The CD4 + T lymphocytes (1 to 3 × 10 5 ) were cultured in a 96-well round-bottom plate with mature autologous DCs previously loaded with pools of peptides (10 mg / ml). The culture is carried out in IMDM medium supplemented with Serum AB (complete IMDM medium) containing 1000U / ml of IL-6 (R&D Systems) and 10ng / ml of IL-12 (R&D Systems). 25 lymphocyte lines were seeded for each peptide pool. The culture is stimulated after one week by adding 10,000 to 30,000 DC loaded with the peptide pools, 20U / ml of IL-2 (R&D Systems) and 10ng / ml of IL-7 (R&D Systems) ). Another stimulation is made after 14 and 21 days of culture. Between 5 and 7 days after the last stimulation, a specificity test is performed by ELISpot. Each culture well constitutes a line of CD4 + T lymphocytes.
4. Evaluation de la spécificité de Lymphocytes T CD4 cultivés avec les peptides par ELISpot 4. Evaluation of the specificity of CD4 T lymphocytes cultured with the peptides by ELISpot
[0113] Des anticorps anti-IFN-g humain (clone 1 -D1 K, Mabtech, Suède) ont été adsorbés à 2,5mg/ml en PBS 1X (Mabtech) sur des plaques 96 puits Multiscreen HA (Millipore). Après une nuit d’incubation à 4°C, les plaques ont été saturées en les incubant 2 heures à 37°C avec du milieu l’IMDM complet. Les lymphocytes T CD4+ ont été incubés pendant 16 heures à 37°C dans ces plaques après avoir été lavés dans du milieu AIM-V contenant 0,5ng/ml d’IL-7, en présence de cellules présentatrices (50000 PBMC/puits) incubés en présence de 10mg/ml de peptides. Après l’incubation, les plaques ont été lavées avec de l’eau distillée, du PBS /Tween 0,05% et enfin du PBS. L’anticorps anti-IFN-y humain 7-B6-1 biotinylé (Mabtech) à 0,25mg/ml en PBS 1X/BSA 1 % a été ajouté dans les plaques (100mL/puits) puis incubé 1 h30 à 37°C. Après plusieurs lavages en PBS ou PBS/tween 0,05%, la sécrétion d’IFN-y a été mise en évidence par l’ajout de l’Extravidine-Phosphatase Alcaline et du substrat NBT/BCIP (Sigma-Aldrich, France). Après 5 à 10 minutes d’incubation, la réaction a été stoppée par lavage à l’eau courante. Après séchage, les spots, reflétant la sécrétion d’IFN- y par chaque lymphocyte T CD4+ activé, ont été comptés sur un lecteur ELISpot (AID). Une lignée de lymphocytes T CD4+ est considérée comme spécifique de l’antigène si le nombre de spots dans les puits contenant l’antigène est au moins deux fois plus élevé que le puits ne contenant pas l’antigène, la différence entre les deux types de puits étant au moins supérieure à 25. Anti-human IFN-g antibodies (clone 1 -D1 K, Mabtech, Sweden) were adsorbed at 2.5 mg / ml in 1X PBS (Mabtech) on 96-well Multiscreen HA plates (Millipore). After overnight incubation at 4 ° C, the plates were saturated by incubating them for 2 hours at 37 ° C with complete IMDM medium. CD4 + T lymphocytes were incubated for 16 hours at 37 ° C in these plates after being washed in AIM-V medium containing 0.5ng / ml IL-7, in the presence of presenting cells (50,000 PBMC / well ) incubated in the presence of 10 mg / ml of peptides. After the incubation, the plates were washed with distilled water, PBS / 0.05% Tween and finally PBS. The biotinylated anti-human IFN-γ antibody 7-B6-1 (Mabtech) at 0.25 mg / ml in 1X PBS / 1% BSA was added to the plates (100mL / well) then incubated for 1 hour 30 minutes at 37 ° C. . After several washes in PBS or PBS / 0.05% tween, the secretion of IFN-γ was demonstrated by the addition of Alkaline Extravidin-Phosphatase and NBT / BCIP substrate (Sigma-Aldrich, France) . After 5 to 10 minutes of incubation, the reaction was stopped by washing with running water. After drying, the spots, reflecting the secretion of IFN- γ by each activated CD4 + T lymphocyte, were counted on an ELISpot reader (AID). A CD4 + T cell line is considered to be antigen specific if the number of spots in the wells containing the antigen is at least twice as high as the well not containing the antigen, the difference between the two types of well being at least greater than 25.
Résultats Results
1. Sélection des peptides par prédiction in silico 1. Selection of peptides by in silico prediction
[0114] Les peptides ont été sélectionnés à l’aide de l’outil de prédiction de liaison au CMH-II, issu de la base de données IEDB (http://www.iedb.org/). Seules les molécules HLA-DR les plus abondamment exprimées à la surface des Cellules Présentatrices d’Antigène (CPA) ont été étudiées. Les allèles DRB1 parmi les plus fréquents dans différentes populations [Tableau 7] ont été sélectionnés. [0114] The peptides were selected using the MHC-II binding prediction tool from the IEDB database (http://www.iedb.org/). Only the HLA-DR molecules most abundantly expressed on the surface of Antigen Presenting Cells (APC) were studied. The DRB1 alleles among the most frequent in different populations [Table 7] were selected.
[0115] Les allèles HLA-DRB3*01 :01 , HLA-DRB3*02:02, HLA-DRB4*01 :01 et HLA-DRB5*01 :01 ont été ajoutées. Ainsi, 15 allèles HLA-DR, représentatifs des populations Caucasienne et Africaine, cités ci-après ont été sélectionnés : HLA- DRB1*01 :01 , HLA-DRB1 *03:01 , HLA-DRB1 *04:01 , HLA-DRB1 *04:05, HLA- DRB1 *07:01 , HLA-DRB1 *08:02, HLA-DRB1 *09:01 , HLA-DRB1*11 :01 , HLA- DRB1*12:01 , HLA-DRB1*13:01 , HLA- DRB1 *15:01 , HLA-DRB3*01 :01 , HLA- DRB3*02:02, HLA-DRB4*01 :01 et HLA-DRB5*01 :01. [0115] The HLA-DRB3 * 01: 01, HLA-DRB3 * 02: 02, HLA-DRB4 * 01: 01 and HLA-DRB5 * 01: 01 alleles were added. Thus, 15 HLA-DR alleles, representative of the Caucasian and African populations, listed below were selected: HLA-DRB1 * 01: 01, HLA-DRB1 * 03: 01, HLA-DRB1 * 04: 01, HLA-DRB1 * 04:05, HLA- DRB1 * 07: 01, HLA-DRB1 * 08: 02, HLA-DRB1 * 09: 01, HLA-DRB1 * 11: 01, HLA- DRB1 * 12: 01, HLA-DRB1 * 13 : 01, HLA- DRB1 * 15: 01, HLA-DRB3 * 01: 01, HLA- DRB3 * 02: 02, HLA-DRB4 * 01: 01 and HLA-DRB5 * 01: 01.
[0116] Les algorithmes NetMHCIIpan et Sturniolo ont permis de prédire la capacité de liaison d’un peptide donné aux molécules HLA de classe II sélectionnées. Pour chaque peptide, un rang est déterminé, son affinité prédite étant comparée à celle de 200000 peptides naturels. Dans le cas présent, les peptides de 9-mers (coeur) avec un rang inférieur à 10% pour au moins 4 allèles HLA-DR, ou les peptides ayant un rang inférieur à 5% pour au moins 1 allèle ont été sélectionnés. Des peptides de 20-mers contenant les coeurs proches ont ensuite été définis, de manière à ce que les coeurs ne soient pas en position 1 ou en position 20 du peptide. 23 peptides 20-mer pour la NP et 33 peptides 20-mer pour la GP ont été sélectionnés pour être testés in vitro. [0117] [Tableau 7] The NetMHCIIpan and Sturniolo algorithms made it possible to predict the binding capacity of a given peptide to selected HLA class II molecules. For each peptide, a rank is determined, its predicted affinity being compared to that of 200,000 natural peptides. In the present case, the 9-mer (core) peptides with a rank of less than 10% for at least 4 HLA-DR alleles, or the peptides with a rank of less than 5% for at least 1 allele were selected. 20-mer peptides containing the close hearts were then defined, so that the hearts were not in position 1 or in position 20 of the peptide. 23 20-mer peptides for NP and 33 20-mer peptides for GP were selected to be tested in vitro. [0117] [Table 7]
Figure imgf000049_0001
Figure imgf000049_0001
[Tableau 71: Fréquence allélique (%) des principales molécules HLA-DRB1 (données issues de www.allefrequencies.net) [Table 71: Allelic frequency (%) of the main HLA-DRB1 molecules (data from www.allefrequencies.net)
Les allèles sélectionnés sont indiqués en gras. 2. Obtention de lignées de lymphocytes T CD4+ spécifiques de la NP et de la GP de ZEBOV Selected alleles are shown in bold. 2. Obtaining CD4 + T lymphocyte lines specific for NP and GP from ZEBOV
[0118] 16 donneurs sains comportant des molécules HLA variées et représentatives des populations caucasiennes et africaines ont été sélectionnés. Des lignées de lymphocytes T CD4+ ont été obtenues par co-culture des lymphocytes T CD4+ avec des cellules dendritiques autologues préalablement chargées avec des pools de peptides. Après amplification des lymphocytes T, chaque lignée a été testée par ELISpot pour sa capacité à reconnaître des pools de peptides puis dans un second test, les peptides individuels contenus dans le pool reconnu par les lignées. Afin de s’assurer de la capacité de chaque donneur à pouvoir répondre, des lignées lymphocytaires CD4+ spécifiques d’un pool de peptides spécifiques du Cytomégalovirus, Epstein-barr et de la grippe ont également été produites. 16 healthy donors comprising various HLA molecules representative of Caucasian and African populations were selected. CD4 + T lymphocyte lines were obtained by co-culture of CD4 + T lymphocytes with autologous dendritic cells previously loaded with pools of peptides. After amplification of the T lymphocytes, each line was tested by ELISpot for its ability to recognize pools of peptides and then in a second test, the individual peptides contained in the pool recognized by the lines. In order to ensure the capacity of each donor to respond, CD4 + lymphocyte lines specific for a pool of peptides specific for Cytomegalovirus, Epstein-barr and influenza were also produced.
[0119] Le [Tableau 8] et le [Tableau 9] présentent les réponses individuelles en lymphocytes T CD4+ spécifiques contre les différents peptides de la NP et de la GP d’Ebola respectivement. Les lignées de lymphocytes T CD4 ont été obtenues par stimulation in vitro par des cellules dendritiques préalablement chargées avec les peptides de la NP ou de la GP. La spécificité pour ces peptides a été évaluée par Elispot IFNy. Le [Tableau 8] et le [Tableau 9] indiquent le taux de répondeurs (en %) et l’intensité de réponse (pourcentage de lignées positives) pour chacun des peptides. [0119] [Table 8] and [Table 9] show the individual responses in specific CD4 + T lymphocytes against the different peptides of Ebola NP and GP respectively. The CD4 T lymphocyte lines were obtained by in vitro stimulation by dendritic cells previously loaded with the NP or GP peptides. The specificity for these peptides was evaluated by Elispot IFNγ. [Table 8] and [Table 9] indicate the rate of responders (in%) and the intensity of response (percentage of positive lines) for each of the peptides.
[0120] [Tableau 8] [0120] [Table 8]
Figure imgf000051_0001
Figure imgf000051_0001
ITableau 81: Réponse individuelle des lymphocytes T CD4 contre les peptides N P [0121] [Tableau 9] Table 81: Individual response of CD4 T lymphocytes against NP peptides [0121] [Table 9]
Figure imgf000052_0001
Figure imgf000052_0001
ITableau 91: Réponse individuelle des lymphocytes T CD4+ contre les peptides GP [0122] Il est à noter que seul un peptide (GP18-37) n’induit de réponse chez aucun des donneurs. Pour chaque peptide, l’intensité de réponse est variable en fonction des donneurs, de 1 à 14 lignées positives. Par ailleurs, le nombre de lignées positives totales par donneur est également extrêmement variable, de 1 à 83 et de 1 à 105 pour la NP et la GP respectivement. Table 91: Individual response of CD4 + T lymphocytes against GP peptides [0122] It should be noted that only one peptide (GP18-37) does not induce a response in any of the donors. For each peptide, the intensity of response is variable depending on the donors, from 1 to 14 positive lines. Furthermore, the number of total positive lines per donor is also extremely variable, from 1 to 83 and from 1 to 105 for PN and GP respectively.
[0123] Les résultats obtenus sont très hétérogènes en fonction des peptides étudiés. Dans le cadre de la vaccination contre le virus Ebola, il est nécessaire de sélectionner les peptides qui permettent d’induire une réponse cellulaire importante chez un maximum d’individus. Les données peuvent donc être analysées selon deux paramètres : le taux (nombre de donneurs répondeurs) et l’intensité (nombre de lignées totales positives) de réponse pour chaque peptide pour les peptides issus de la NP et la GP respectivement. Dix peptides pour la NP et 8 pour la GP induisent des réponses chez 50% des donneurs ou plus. On peut noter une bonne corrélation entre taux et intensité de réponse, en particulier pour la NP. En effet, les peptides répondant chez le plus grand nombre de donneurs sont également ceux pour lesquels le nombre de lignées positives est le plus élevé. Ces peptides peuvent donc permettre une bonne couverture de la population, tout en induisant une réponse efficace. The results obtained are very heterogeneous depending on the peptides studied. In the context of vaccination against the Ebola virus, it is necessary to select the peptides which make it possible to induce a significant cellular response in as many individuals as possible. The data can therefore be analyzed according to two parameters: the rate (number of responder donors) and the intensity (number of total positive lines) of response for each peptide for peptides derived from NP and GP respectively. Ten peptides for NP and 8 for GP induce responses in 50% or more of donors. We can note a good correlation between response rate and intensity, in particular for PN. Indeed, the peptides responding in the largest number of donors are also those for which the number of positive lines is the highest. These peptides can therefore provide good coverage of the population, while inducing an effective response.
[0124] Les peptides identifiés et étudiés sont issus de la souche Zaïre du virus[0124] The peptides identified and studied are derived from the Zaire strain of the virus
Ebola. La conservation de la séquence peptidique entre les différentes souches du virus Ebola a été étudiée pour les peptides présentant un fort taux de réponse. Les résultats sont représentés en pourcentage d’identité de séquence par rapport à la souche Zaïre. Le pourcentage d’identité des peptides entre les différentes souches est plus élevé pour les peptides de la NP que de la GP [Tableau 10] et [Tableau 11] Ces peptides, en particulier les peptides NP, devraient ainsi pouvoir être utilisés dans une composition vaccinale pour l’ensemble des souches du virus Ebola. [0125] [Tableau 10] Ebola. The conservation of the peptide sequence between the different strains of the Ebola virus has been studied for peptides exhibiting a high response rate. The results are represented as a percentage of sequence identity relative to the Zaire strain. The percentage identity of the peptides between the different strains is higher for the NP peptides than for the GP [Table 10] and [Table 11] These peptides, in particular the NP peptides, should thus be able to be used in a composition. vaccine for all strains of the Ebola virus. [0125] [Table 10]
Figure imgf000054_0001
Figure imgf000054_0001
[Tableau 10]: Conservation des séquences des peptides issus de la NP entre les différentes souches du virus Ebola [Table 10]: Conservation of the sequences of the peptides derived from NP between the different strains of the Ebola virus
[0126] [Tableau 11] [0126] [Table 11]
Figure imgf000054_0002
Figure imgf000054_0002
[Tableau 11] : Conservation des séquences des peptides issus de la GP entre les différentes souches du virus Ebola [Table 11]: Conservation of the sequences of the peptides derived from GP between the different strains of the Ebola virus
3. Combinaison de peptides en longs fragments d’intérêt 3. Combination of peptides into long fragments of interest
[0127] La couverture de différentes populations est un des enjeux majeurs dans le développement d’un vaccin contre le virus Ebola. Compte tenu du rôle majeur des lymphocytes T CD4+ dans l’initiation des réponses humorales et cytotoxiques, il est avantageux de combiner les épitopes T CD8 d’Ebola (exemple 1 ) avec les épitopes T CD4 d’Ebola identifiés ci-dessus pour augmenter l’efficacité d’un vaccin contre le virus Ebola. En outre, il est nécessaire de déterminer un cocktail minimal d’épitopes T CD4 et d’épitopes T CD8 permettant d’obtenir une couverture maximale de la population à vacciner [0127] The coverage of different populations is one of the major issues in the development of a vaccine against the Ebola virus. Given the major role of CD4 + T lymphocytes in the initiation of humoral and cytotoxic responses, it is advantageous to combine the CD8 T epitopes from Ebola (example 1) with the CD4 T epitopes from Ebola identified above to increase the effectiveness of an Ebola virus vaccine. In addition, it is necessary to determine a minimal cocktail CD4 T epitopes and CD8 T epitopes to achieve maximum coverage of the population to be vaccinated
Dans ce cas, 6 LSP pour la NP peuvent ainsi être établis ([Tableau 12]). In this case, 6 LSPs for PN can thus be established ([Table 12]).
[0128] [Tableau 12] [0128] [Table 12]
Figure imgf000055_0001
Figure imgf000055_0001
[Tableau 12] : LSP issus de la NP contenant des épitopes T CD4 et T CD8 [0129] L’objectif est de déterminer un jeu de peptides permettant une couverture maximale des donneurs testés, aussi bien en réponse T CD4 qu’en réponse T CD8. Avec cette combinaison, la couverture maximale est obtenue aussi bien pour les T CD4 comme pour les T CD8. [Table 12]: PSL from NP containing T CD4 and T CD8 epitopes The objective is to determine a set of peptides allowing maximum coverage of the donors tested, both in T CD4 response and T CD8 response. With this combination, maximum coverage is obtained for both T CD4 and T CD8.
[0130] Pour la GP, 6 LSP peuvent également être combinés ([Tableau 13]). [0130] For the GP, 6 LSPs can also be combined ([Table 13]).
[0131] [Tableau 13] [0131] [Table 13]
Figure imgf000056_0001
Figure imgf000056_0001
[Tableau 13] : LSP issus de la GP contenant des épitopes T CD4 et T CD8 [0132] Les 6 LSP de la GP sélectionnés ne permettent pas de couvrir l’allèle HLA-B*08, aucun épitope n’ayant été obtenu pour cet allèle. Les allèles HLA-A*11 et HLA-B*35 ne sont également pas couverts par les LSP, les épitopes obtenus étant trop éloignés. Afin d’obtenir la couverture optimale, il faut alors ajouter le peptide GP664-673 (épitope HLA-A*11 ). Seuls 14 des 15 donneurs sont couverts par cette combinaison, tandis que la couverture est maximale pour les donneurs CD4+. [Table 13]: PSL from GP containing T CD4 and T CD8 epitopes The 6 LSPs of the GP selected do not make it possible to cover the HLA-B * 08 allele, no epitope having been obtained for this allele. The HLA-A * 11 and HLA-B * 35 alleles are also not covered by the LSPs, the epitopes obtained being too far apart. In order to obtain optimal coverage, the peptide GP664-673 (epitope HLA-A * 11) must then be added. Only 14 of the 15 donors are covered by this combination, while coverage is maximum for CD4 + donors.
Conclusion Conclusion
[0133] Cette étude a permis de mettre en évidence des épitopes T CD8 immunogènes issus de la NP et de la GP du virus Ebola, capables de stimuler les lymphocytes T CD8, dans une cohorte de donneurs sains représentative des populations caucasiennes et africaines (groupe de référence). This study made it possible to demonstrate immunogenic CD8 T epitopes derived from NP and GP of the Ebola virus, capable of stimulating CD8 T lymphocytes, in a cohort of healthy donors representative of the Caucasian and African populations (group reference).
[0134] La NP est plus immunogène que la GP, 54 épitopes ayant été obtenus pour la première, et 28 pour la seconde. Ces résultats sont cohérents avec les observations faites par Mc Elroy et al., (Mc Elroy et al., 2015, précité) chez des patients ayant été infectés par le virus. Il a en effet observé qu’il existait une réponse lymphocytaire T CD4+ et CD8+ contre les différentes protéines virales, et en particulier contre la NP. NP is more immunogenic than GP, 54 epitopes having been obtained for the first, and 28 for the second. These results are consistent with the observations made by Mc Elroy et al. (Mc Elroy et al., 2015, cited above) in patients who have been infected with the virus. He observed that there was a CD4 + and CD8 + T lymphocyte response against the various viral proteins, and in particular against PN.
[0135] Aucun épitope restreint à l’allèle HLA-A*23 (prépondérant dans les populations africaines) n’a été obtenu. Néanmoins, la combinaison des différents épitopes permet d’obtenir une couverture vaccinale potentielle des populations africaines et caucasiennes entre 68 et 98%. [0135] No epitope restricted to the HLA-A * 23 allele (predominant in African populations) was obtained. Nevertheless, the combination of the different epitopes makes it possible to obtain a potential vaccination coverage of African and Caucasian populations between 68 and 98%.
[0136] Ces épitopes T CD8 d’Ebola sont avantageusement combinés avec les épitopes T CD4 d’Ebola (exemple 2) de façon à obtenir une combinaison de peptides permettant une couverture maximale des donneurs testés, aussi bien en réponse T CD4 qu’en réponse T CD8 dirigée contre le virus Ebola. La combinaison des épitopes T CD4 et T CD8 d’Ebola permet ainsi d’augmenter l’efficacité d’un vaccin contre le virus Ebola. These Ebola CD8 T epitopes are advantageously combined with the Ebola CD4 T epitopes (example 2) so as to obtain a combination of peptides allowing maximum coverage of the donors tested, both in T CD4 response and in CD8 T response against Ebola virus. The combination of the T CD4 and T CD8 epitopes of Ebola thus makes it possible to increase the effectiveness of a vaccine against the Ebola virus.

Claims

Revendications Claims
1. Composition immunogène ou vaccinale contre le virus Ebola, comprenant au moins une combinaison d’au moins 10 épitopes T CD8 du virus Ebola qui est apte à être présentée par au moins 13 molécules HLA I prépondérantes différentes choisies parmi HLA-A1 , HLA-A2, HLA-A3, HLA-A11 , HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA-B40, HLA-B44 et HLA-B51 et à induire une réponse en lymphocytes T CD8+ humains spécifiques chez des individus exprimant au moins l’une desdites molécules HLA-I, et lesdits épitopes étant sélectionnés dans le groupe constitué par : 1. Immunogenic or vaccine composition against the Ebola virus, comprising at least a combination of at least 10 CD8 T epitopes of the Ebola virus which is capable of being presented by at least 13 different predominant HLA I molecules chosen from HLA-A1, HLA- A2, HLA-A3, HLA-A11, HLA-A24, HLA-A29, HLA-B7, HLA-B8, HLA-B15, HLA-B18, HLA-B35, HLA-B40, HLA-B44 and HLA-B51 and inducing a response in specific human CD8 + T lymphocytes in individuals expressing at least one of said HLA-I molecules, and said epitopes being selected from the group consisting of:
- les épitopes SEQ ID NO : 28 et 46 de la nucléoprotéine (NP), restreints à - the epitopes SEQ ID NO: 28 and 46 of the nucleoprotein (NP), restricted to
HLA-A1 ; HLA-A1;
- les épitopes SEQ ID NO : 2, 5, 7, 8, 11 , 13, 14, 17, 20, 23, 36, 38 et 45 de la NP et SEQ ID NO : 53, 57, 58, 63, 64, 65, 72 et 73 de la glycoprotéine (GP), restreints à HLA-A2 ; - the epitopes SEQ ID NO: 2, 5, 7, 8, 11, 13, 14, 17, 20, 23, 36, 38 and 45 of NP and SEQ ID NO: 53, 57, 58, 63, 64, 65, 72 and 73 of the glycoprotein (GP), restricted to HLA-A2;
. - les épitopes SEQ ID NO : 9 et 44 de la NP et SEQ ID NO : 69 de la GP, restreints à HLA-A3 ; . the epitopes SEQ ID NO: 9 and 44 of the NP and SEQ ID NO: 69 of the GP, restricted to HLA-A3;
- les épitopes SEQ ID NO : 24 de la NP et SEQ ID NO : 71 de la GP, restreints à HLA-A11 ; the epitopes SEQ ID NO: 24 of the NP and SEQ ID NO: 71 of the GP, restricted to HLA-A11;
- les épitopes SEQ ID NO : 16 de la NP et SEQ ID NO : 50 et 51 de la GP restreints à HLA-A24 ; the epitopes SEQ ID NO: 16 of NP and SEQ ID NO: 50 and 51 of GP restricted to HLA-A24;
- les épitopes SEQ ID NO : 10 et 41 de la NP, restreints à HLA-A29 ; - the epitopes SEQ ID NO: 10 and 41 of NP, restricted to HLA-A29;
- les épitopes SEQ ID NO : 33 de la NP, restreint à HLA-B7 ; - the epitopes SEQ ID NO: 33 of NP, restricted to HLA-B7;
- les épitopes SEQ ID NO : 26 de la NP, restreint à HLA-B8 ; - the epitopes SEQ ID NO: 26 of NP, restricted to HLA-B8;
- les épitopes SEQ ID NO : 12, 15, 22, 29 et 37 de la NP et SEQ ID NO : 52 de la GP, restreints à HLA-B15 ; the epitopes SEQ ID NO: 12, 15, 22, 29 and 37 of the NP and SEQ ID NO: 52 of the GP, restricted to HLA-B15;
- l’épitope SEQ ID NO : 25 de la NP, restreint à HLA-B18 ; - the epitope SEQ ID NO: 25 of NP, restricted to HLA-B18;
- les épitopes SEQ ID NO : 19, 40, 43, 48 de la NP et SEQ ID NO : 56 de la GP, restreints à HLA-B35 ; the epitopes SEQ ID NO: 19, 40, 43, 48 of the NP and SEQ ID NO: 56 of the GP, restricted to HLA-B35;
- les épitopes SEQ ID NO : 3, 30, 39 et 47 de la NP et SEQ ID NO : 60 et 68 de la GP, restreints à HLA-B40 ; - the epitopes SEQ ID NO: 3, 30, 39 and 47 of NP and SEQ ID NO: 60 and 68 of GP, restricted to HLA-B40;
- les épitopes SEQ ID NO : 4 et 31 de la NP et SEQ ID NO : 59, 62, 66 et 67 de la GP, restreints à HLA-B44 ; - les épitopes SEQ ID NO : 18 et 32 de la NP et SEQ ID NO : 70 de la GP, restreints à HLA-B51 ; the epitopes SEQ ID NO: 4 and 31 of NP and SEQ ID NO: 59, 62, 66 and 67 of GP, restricted to HLA-B44; the epitopes SEQ ID NO: 18 and 32 of the NP and SEQ ID NO: 70 of the GP, restricted to HLA-B51;
- l’épitope SEQ ID NO : 61 de la GP, restreint à HLA-A1 et HLA-A29 ; - the epitope SEQ ID NO: 61 of GP, restricted to HLA-A1 and HLA-A29;
- l’épitope SEQ ID NO : 21 de la NP, restreint à HLA-A2 et HLA-A24 ; - the epitope SEQ ID NO: 21 of NP, restricted to HLA-A2 and HLA-A24;
- l’épitope SEQ ID NO : 6 de la NP, restreint à HLA-A29 et HLA-B15 ; - the epitope SEQ ID NO: 6 of NP, restricted to HLA-A29 and HLA-B15;
- l’épitope SEQ ID NO : 35 de la NP, restreint à HLA-B7 et HLA-B35 ; - the epitope SEQ ID NO: 35 of NP, restricted to HLA-B7 and HLA-B35;
- - l’épitope SEQ ID NO : 27 de la NP, restreint à HLA-B7 et HLA-B51 ;- - the epitope SEQ ID NO: 27 of NP, restricted to HLA-B7 and HLA-B51;
- l’épitope SEQ ID NO : 55 de la GP, restreint à HLA-B7, HLA-B18 et HLA-- the epitope SEQ ID NO: 55 of GP, restricted to HLA-B7, HLA-B18 and HLA-
B51 ; B51;
- les épitopes SEQ ID NO : 34 et 42 de la NP, restreints à HLA-B18 et HLA-B40 ; et - the epitopes SEQ ID NO: 34 and 42 of NP, restricted to HLA-B18 and HLA-B40; and
- l’épitope SEQ ID NO : 54 de la GP, restreint à HLA-B40 et HLA-B44. - the epitope SEQ ID NO: 54 of GP, restricted to HLA-B40 and HLA-B44.
2. Composition immunogène ou vaccinale selon la revendication 1 , comprenant une combinaison de 10 à 25 épitopes apte à être présentée par au moins 13 ou la totalité desdites molécules HLA I prépondérantes. 2. Immunogenic or vaccine composition according to claim 1, comprising a combination of 10 to 25 epitopes capable of being presented by at least 13 or all of said predominant HLA I molecules.
3. Composition immunogène ou vaccinale selon la revendication 1 ou 2, comprenant une combinaison d’épitopes sélectionnés dans le groupe constitué par : les séquences SEQ ID NO : 3 à 7, 9 à 22, 24 à 35 et 37 à 48 de la NP et les séquences SEQ ID NO : 50 à 52, 54 à 56 et 59 à 64 et 66 à 71 de la GP. 3. Immunogenic or vaccine composition according to claim 1 or 2, comprising a combination of epitopes selected from the group consisting of: the sequences SEQ ID NO: 3 to 7, 9 to 22, 24 to 35 and 37 to 48 of the NP. and the sequences SEQ ID NO: 50 to 52, 54 to 56 and 59 to 64 and 66 to 71 of the GP.
4. Composition immunogène ou vaccinale selon la revendication 3, comprenant au moins un épitope sélectionné dans le groupe constitué par : les séquences SEQ ID NO : 5, 6, 10, 15, 17, 24, 25, 34, 41 et 42 de la NP et les séquences SEQ ID NO : 55, 61 et 71 de la GP. 4. Immunogenic or vaccine composition according to claim 3, comprising at least one epitope selected from the group consisting of: the sequences SEQ ID NO: 5, 6, 10, 15, 17, 24, 25, 34, 41 and 42 of the NP and the sequences SEQ ID NO: 55, 61 and 71 of the GP.
5. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 4, comprenant au moins un épitope T CD8 conservé dans différentes souches du virus Ebola, choisi parmi les séquences SEQ ID NO : 3 à 8, 10 à 39, 41 , 43, 47 et 48 de la NP et les séquences SEQ ID NO : 51 à 60 et 67 à 71 de la GP; de préférence choisi parmi les séquences SEQ ID NO : 5, 6, 12, 15 à 24, 27 à 38 et 41 de la NP et les séquences SEQ ID NO : 51 , 54, 55, 57 à 60, 67 et 70 de la GP ; de manière encore plus préférée parmi les séquences SEQ ID NO : SEQ ID NO : 15 à 18, 20 à 24, 27, 30 à 33, 36 et 37 de la NP et les séquences SEQ ID NO : 55, 57, 58, 60 et 67 de la GP. 5. Immunogenic or vaccine composition according to any one of claims 1 to 4, comprising at least one CD8 T epitope conserved in different strains of the Ebola virus, chosen from the sequences SEQ ID NO: 3 to 8, 10 to 39, 41, 43, 47 and 48 of the NP and the sequences SEQ ID NO: 51 to 60 and 67 to 71 of the GP; preferably chosen from the sequences SEQ ID NO: 5, 6, 12, 15 to 24, 27 to 38 and 41 of the NP and the sequences SEQ ID NO: 51, 54, 55, 57 to 60, 67 and 70 of the GP; even more preferably among the sequences SEQ ID NO: SEQ ID NO: 15 to 18, 20 to 24, 27, 30 to 33, 36 and 37 of the NP and the sequences SEQ ID NO: 55, 57, 58, 60 and 67 of the GP.
6. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 5, comprenant une combinaison d’épitopes T CD8 de la NP ou une combinaison d’épitopes T CD8 de la NP et de la GP. 6. An immunogenic or vaccine composition according to any one of claims 1 to 5, comprising a combination of CD8 T epitopes from NP or a combination of CD8 T epitopes from NP and GP.
7. Composition immunogène ou vaccinale selon la revendication 6, comprenant une combinaison d’épitopes T CD8 de la NP comprenant au moins : 7. Immunogenic or vaccine composition according to claim 6, comprising a combination of CD8 T epitopes of NP comprising at least:
a) les épitopes SEQ ID NO : 5 restreint à HLA-A2 ; SEQ ID NO : 16 restreint à HLA-A24, SEQ ID NO : 26 restreint à HLA-B8, SEQ ID NO : 28 restreint à HLA-A1 et SEQ ID NO : 31 restreint à HLA-B44 ; a) the epitopes SEQ ID NO: 5 restricted to HLA-A2; SEQ ID NO: 16 restricted to HLA-A24, SEQ ID NO: 26 restricted to HLA-B8, SEQ ID NO: 28 restricted to HLA-A1 and SEQ ID NO: 31 restricted to HLA-B44;
b) l’un des épitopes SEQ ID NO : 13, SEQ ID NO : 14 ou SEQ ID NO : 17 restreint à HLA-A2 , de préférence l’épitope SEQ ID NO : 17; b) one of the epitopes SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 17 restricted to HLA-A2, preferably the epitope SEQ ID NO: 17;
c) l’un des épitopes SEQ ID NO : 9 ou SEQ ID NO : 44 restreint à HLA- A3 ; de préférence l’épitope SEQ ID NO : 44 ; c) one of the epitopes SEQ ID NO: 9 or SEQ ID NO: 44 restricted to HLA-A3; preferably the epitope SEQ ID NO: 44;
d) l’épitope SEQ ID NO : 24 restreint à HLA-A11 ; d) the epitope SEQ ID NO: 24 restricted to HLA-A11;
e) l’épitope SEQ ID NO : 6 restreint à HLA-A29 et HLA-B15 ou l’un des épitopes SEQ ID NO : 10 ou SEQ ID NO : 41 restreint à HLA-A29 ; de préférence l’épitope SEQ ID NO : 6; e) the epitope SEQ ID NO: 6 restricted to HLA-A29 and HLA-B15 or one of the epitopes SEQ ID NO: 10 or SEQ ID NO: 41 restricted to HLA-A29; preferably the epitope SEQ ID NO: 6;
f) l’un des épitopes SEQ ID NO : 3, SEQ ID NO : 30, SEQ ID NO : 39 ou SEQ ID NO : 47 restreint à HLA-B40 ou l’un des épitopes SEQ ID NO : 34 ou SEQ ID NO : 42 restreints à HLA-B40 et HLA-B18, de préférence l’épitope SEQ ID NO : 30, SEQ ID NO : 34 ou SEQ ID NO : 42, de manière plus préférée l’épitope SEQ ID NO : 34 ou SEQ ID NO : 42 et ; f) one of the epitopes SEQ ID NO: 3, SEQ ID NO: 30, SEQ ID NO: 39 or SEQ ID NO: 47 restricted to HLA-B40 or one of the epitopes SEQ ID NO: 34 or SEQ ID NO : 42 restricted to HLA-B40 and HLA-B18, preferably the epitope SEQ ID NO: 30, SEQ ID NO: 34 or SEQ ID NO: 42, more preferably the epitope SEQ ID NO: 34 or SEQ ID NO: 42 and;
g) l’épitope SEQ ID NO : 27 restreint à HLA-B7 et HLA-B51 , l’épitope SEQ ID NO : 33 restreint à HLA-B7 ou l’épitope SEQ ID NO : 35 restreint à HLA-B7 et HLA-B35 , de préférence l’épitope SEQ ID NO : 27 ou SEQ ID NO : 35 ; de manière plus préférée l’épitope SEQ ID NO : 27 . g) the epitope SEQ ID NO: 27 restricted to HLA-B7 and HLA-B51, the epitope SEQ ID NO: 33 restricted to HLA-B7 or the epitope SEQ ID NO: 35 restricted to HLA-B7 and HLA- B35, preferably the epitope SEQ ID NO: 27 or SEQ ID NO: 35; more preferably the epitope SEQ ID NO: 27.
8. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 5, comprenant une combinaison d’épitopes T CD8 de la GP comprenant au moins 8. Immunogenic or vaccine composition according to any one of claims 1 to 5, comprising a combination of CD8 T epitopes of GP comprising at least
- l’épitope SEQ ID NO : 61 restreint à HLA-A1 et HLA-A29 ; - l’épitope SEQ ID NO : 63 ou SEQ ID NO : 64 restreint à HLA-A2 ; the epitope SEQ ID NO: 61 restricted to HLA-A1 and HLA-A29; the epitope SEQ ID NO: 63 or SEQ ID NO: 64 restricted to HLA-A2;
- l’épitope SEQ ID NO : 69 restreint à HLA-A3 ; - the epitope SEQ ID NO: 69 restricted to HLA-A3;
- l’épitope SEQ ID NO : 71 restreint à HLA-A11 ; - the epitope SEQ ID NO: 71 restricted to HLA-A11;
- l’épitope SEQ ID NO : 50 ou SEQ ID NO : 51 restreint à HLA-A24 ; - the epitope SEQ ID NO: 50 or SEQ ID NO: 51 restricted to HLA-A24;
- l’épitope SEQ ID NO : 55 restreint à HLA-B7 , HLA-B18 et HLA-B51 ; , - the epitope SEQ ID NO: 55 restricted to HLA-B7, HLA-B18 and HLA-B51; ,
- l’épitope SEQ ID NO : 52 restreint à HLA-B15 ; - the epitope SEQ ID NO: 52 restricted to HLA-B15;
- l’épitope SEQ ID NO : 56 restreint à HLA-B35 ; - the epitope SEQ ID NO: 56 restricted to HLA-B35;
- l’épitope SEQ ID NO : 54 restreint à HLA-B40 et HLA-B44, et ; - the epitope SEQ ID NO: 54 restricted to HLA-B40 and HLA-B44, and;
- l’épitope SEQ ID NO : 59 ou SEQ ID NO : 66 restreint à HLA-B44. - the epitope SEQ ID NO: 59 or SEQ ID NO: 66 restricted to HLA-B44.
9. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 8, comprenant en outre une séquence choisie parmi les séquences SEQ ID NO : 74 à 96 et 129 à 138 de la NP et les séquences SEQ ID NO : 97 à 128 et 139 à 148 de la GP. 9. Immunogenic or vaccine composition according to any one of claims 1 to 8, further comprising a sequence chosen from the sequences SEQ ID NO: 74 to 96 and 129 to 138 of the NP and the sequences SEQ ID NO: 97 to 128. and 139 to 148 of the GP.
10. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 9, comprenant un ou plusieurs peptides issus de la protéine NP de séquence SEQ ID NO : 1 ou GP de séquence SEQ ID NO : 49 ou vecteurs recombinants codant le ou lesdits peptides, polypeptides et/ou protéines. 10. Immunogenic or vaccine composition according to any one of claims 1 to 9, comprising one or more peptides derived from the NP protein of sequence SEQ ID NO: 1 or GP of sequence SEQ ID NO: 49 or recombinant vectors encoding said (s). peptides, polypeptides and / or proteins.
11. Composition immunogène ou vaccinale selon la revendication 10, comprenant les peptides NP de séquence SEQ ID NO : 80, 135, 149, 150, 151 et 152 ou les peptides GP de séquence SEQ ID NO : 71 , 107, 148, 153, 154, 155 et 156. 11. Immunogenic or vaccine composition according to claim 10, comprising the NP peptides of sequence SEQ ID NO: 80, 135, 149, 150, 151 and 152 or the GP peptides of sequence SEQ ID NO: 71, 107, 148, 153, 154, 155 and 156.
12. Composition immunogène ou vaccinale selon l’une quelconque des revendications 1 à 11 , pour son utilisation comme vaccin dans la prévention d’une infection par le virus Ebola. 12. Immunogenic or vaccine composition according to any one of claims 1 to 11, for its use as a vaccine in the prevention of infection with the Ebola virus.
13. Utilisation in vitro d’une composition selon l’une quelconque des revendications 1 à 11 , pour le diagnostic d’une infection par le virus Ebola ou l’immunomonitorage de la réponse cellulaire contre le virus Ebola. 13. In vitro use of a composition according to any one of claims 1 to 11, for the diagnosis of an infection by the Ebola virus or the immunomonitoring of the cellular response against the Ebola virus.
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