Fine-tuning of the immune system

Abstract

The present invention provides a CD1 peptide epitope for treating a disease caused by an intracellular pathogen, a method for identifying such a CD1 peptide epitope, a pharmaceutical kit comprising such a CD1 peptide epitope, and a method for treating a patient for a disease caused by an intracellular pathogen or preventing such an infection on the basis of vaccination with the CD1 peptide epitope.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the immunology field for the specific treatment of intracellular pathogens after a strong cytotoxic response dominated by cytotoxic T cells. BACKGROUND

[0002] Intracellular pathogens, such as viruses or bacteria, represent a major concern, as there is no real strong specific solution.

[0003] Indeed, the response to viral infection requires the activation of a strong cytotoxic component dominated by CD8 lineage T cells and natural killer (NK) cells. This is naturally obtained by the recognition of membrane-bound and cell membrane-distributed pathogen-associated molecular patterns (PAMPS) by some pattern recognition receptors (PPR) of the host, including toll-like receptors (TLRs), RIG-like receptors (RIGs) and Nod-like receptors (NLRs). Subsequently, various signaling pathways involving NF-kB and IRF are activated, which finally produce cytokines, in particular type I and III interferons.

[0004] However, in practice, each single step in these activation pathways can be used to implement targets for viral interference or proteolysis, thus weakening the innate and cytotoxic immune response. After infection, the pathogen should immediately avoid the immune system, the pathogen is resistant, either the immune system does not see the pathogen or the pathogen is able to modulate the host cell to avoid the immune system.

[0005] Chemotherapy, including treatment with potent antibiotics and antiviral molecules, represents a solution, but has significant side effects and often fails to kill all the pathogens hidden in host cells or in tissues as reservoirs, which means that, when treatment has to be stopped, for example to avoid these side effects to the patient, the pathogen grows again and infects other cells or tissues of the patient.

[0006] Even prophylactic or therapeutic (therapeutic) vaccination is not sufficient to induce the correct immune response to all intracellular pathogens: sometimes it can be possible to develop a vaccine, sometimes all previous attempts have failed.

[0007] Vaccination strategies usually exploit viral or pathogen-derived proteins, or their encoded DNA or RNA sequences, administered with adjuvants. This vaccination promotes the production of specific antibodies preferentially in the context of CD4+ T cell expansion, but hardly produces any cytotoxicity.

[0008] In addition to this intrinsic limitation, the strength of the response to a vaccine also depends on the ability of MHC class II determinants to present viral or pathogen epitopes to CD4+ T cells. The apparent polymorphism of MHC class II determinants in the population makes the efficacy of CD4+ T cell activation vary greatly from one individual to another, which adds another uncertainty in predicting the efficacy of a vaccine and in inducing memory. In fact, it is a common observation to see the antibody response to a virus disappear over time.

[0009] Therefore, there is an urgent need to develop vaccination strategies to circumvent as much as possible the preferential outgrowth of CD4+ T cells at the expense of cytotoxic cells, and to cope with the intrinsic variability of the response due to MHC class II polymorphism.

[0010] On the other hand, the ability of CD1 molecules to load peptide epitopes, in particular those bearing hydrophobic motives at specific positions, has been discovered by the inventors of the present patent application and developed for therapeutic applications, for example in patent applications WO 2012 / 069572, WO 2012 / 069575, WO 2013 / 174805 and WO 2018 / 189405.

[0011] However, direct approaches based on CD1 have not yet been widely recognized as a tool in immunology. Indeed, the main purpose of these patent documents is to modify such peptide epitopes to specifically modulate immune responses, rather than directly using their native form for vaccination purposes and / or training the immune system. SUMMARY

[0012] The present invention relates to a CD1 peptide epitope for use in the prevention or treatment of a disease caused by an intracellular pathogen, wherein:

[0013] (i) said disease is preferably a viral disease caused by a virus selected from the group consisting of Flaviviridae, Coronaviridae, Orthomyxoviridae, Herpesviridae and Picornaviridae,

[0014] (ii) said CD1 peptide epitope is a fragment of a protein expressed by said intracellular pathogen, wherein said CD1 peptide epitope comprises the sequence X1X2X3X4X5X6X7, wherein said X2, X3, X4, X5 and X6 independently represent any amino acid, and said X1 and X7 residues are independently {F; W; T; H or Y}, and

[0015] (iii) said use comprises the administration of an inactivated form of said intracellular pathogen or of a second peptide based on a protein of said intracellular pathogen, wherein said inactivated intracellular pathogen or said second peptide is administered after the administration of the CD1 peptide epitope, and wherein said second peptide comprises the CD1 epitope sequence defined in (ii) and at least one MHC class I epitope.

[0016] Further, the present application relates to an (in vitro) method for identifying one or several (plurality of) peptide epitopes to activate Thl-like (IFNy and / or IL-12) NKT cells and / or cytotoxic responses to an intracellular pathogen, said method comprising the steps of: identifying CD1 peptide epitopes in said intracellular pathogen, and (in vitro) measuring the ability of the identified CD1 peptide epitopes to activate Thl-like NKT cells and / or cytotoxic responses.

[0017] Preferably, in this (in vitro) method, the activation of NKT cells is measured by incubating the peptide epitopes with cells expressing CD1 molecules at their surface, followed by the addition of a population of NKT cells and determining the activation of said NKT cells.

[0018] Preferably, in this (in vitro) method, the identification of CD1 peptide epitopes is measured by the ability of peptides (or fragments of peptides) of the intracellular pathogen to bind to this CD1 molecule.

[0019] Preferably, in this (in vitro) method, the identified CD1 peptide epitopes have the sequence X1X2X3X4X5X6X7, wherein the X2X3X4X5X6 independently represent any amino acid, wherein the X1and X7residues are independently {F; W; T; H or Y}, wherein, preferably, X4 is {I; V; L or M} and / or wherein, preferably, at least one X1or X7residue is {F; W or Y} and / or wherein, preferably, the X1X2X3X4X5X6X7sequence belongs to and / or forms an alpha-helix, possibly after binding to CD1.

[0020] Advantageously, in this (in vitro) method, the identified CD1 peptide epitopes do not comprise MHC class II epitopes.

[0021] Preferably, in this (in vitro) method, the activation of NKT cells is determined by quantifying interferon (IFN)-y and / or interleukin (IL)-12 levels, possibly IL-4, IFNa and / or IFNp are also quantified, and the ratio IFNy / IL-4 (or IL-12 / IL-4) is measured; an increase means a cytotoxic response.

[0022] A related aspect of the present application is a CD1 peptide epitope for use in the prevention or treatment of a disease, preferably a disease caused by an intracellular pathogen, wherein the disease is preferably a viral disease, more preferably from the group consisting of flaviviruses, coronaviruses, orthomyxoviruses, herpesviruses and picornaviruses, and wherein the CD1 peptide epitope is a fragment of a protein expressed by the intracellular pathogen, advantageously the fragment does not comprise MHC class II epitopes.

[0023] Advantageously, the CD1 peptide epitope (for preventing or treating a disease caused by an intracellular pathogen) can be obtained by the above (in vitro) method.

[0024] Another related aspect of the application is a kit (for treating a disease, preferably a disease caused by an intracellular pathogen), comprising a first peptide, which is the above-mentioned CD1 peptide epitope (advantageously not comprising an MHC class II epitope), and a second peptide, which is from the intracellular pathogen, and which is a peptide comprising the first peptide epitope in addition to other epitopes (advantageously at least one MHC class II epitope).

[0025] The kit is preferably for treating a viral disease, more preferably from the group consisting of flaviviruses, coronaviruses, orthomyxoviruses, herpesviruses and picornaviruses.

[0026] Preferably, the kit further comprises an adjuvant for vaccination with the first peptide (CD1 peptide epitope, advantageously not comprising an MHC class II epitope) and / or an adjuvant for vaccination with the second peptide (advantageously comprising a CD1 peptide epitope and an MHC class II epitope, possibly also comprising other MHC class I or II epitopes).

[0027] Preferably, the kit further comprises means for quantifying IgG1 and / or IgG3 (or total IgG1 and / or IgG3) specific for the CD1 peptide epitope, and / or means for quantifying all IgG (or at least IgG2), or IgG (or total IgG or IgG other than IgG1 and / or IgG3, such as IgG2) other than IgG1 and / or IgG3 specific for the CD1 peptide epitope.

[0028] Preferably, the kit further comprises means for quantifying IgG1 and / or IgG3 (or total IgG1 and / or IgG3) specific for the second peptide, and / or means for quantifying all IgG (or IgG2) specific for the second peptide, or all IgG (or IgG2) other than IgG1 and / or IgG3 specific for the second peptide (or total IgG, or IgG other than IgG1 and / or IgG3, such as IgG2), which IgG (or IgG2) other than IgG1 and IgG3 can be non-specific for the (first) CD1 peptide epitope.

[0029] Advantageously, the ratio of {IgG1 and / or IgG3} : IgG (or IgG2), and / or the (relative) increase in (specific) IgG1 and / or IgG3, is indicative of a cytotoxic response of the immune system, which reflects a correct response to the treatment.

[0030] Preferably, the kit further comprises means to quantify the levels of IFNγ and / or IL-12 and / or IL-4 and / or IFNα and / or IFNβ.

[0031] Another related aspect of the application is a pharmaceutical method for modulating the immune system of a patient to generate a specific Thl-like and / or cytotoxic response against an intracellular pathogen. The pharmaceutical method comprises the steps of identifying a CD1 peptide epitope in a protein expressed by said intracellular pathogen, and (in vitro) measuring the identified CD1 peptide epitope to activate a Thl-like (IFNγ and / or IL-12) NKT cell response, and / or to induce a cytotoxic response, and administering to the patient a first peptide, preferably together with a vaccine adjuvant, which first peptide is (substantially) the CD1 peptide epitope.

[0032] Preferably, in the method, the step of identifying a CD1 peptide epitope in an intracellular pathogen comprises determining the ability of the peptide to bind to said CD1 molecule.

[0033] Preferably, the method further comprises incubating the peptide epitope with CD1 -bearing cells, followed by the addition of a population of NKT cells, and determining the activation of these NKT cells.

[0034] Preferably, in the method, the (cytotoxic) activation of the NKT cells is determined by quantification of the levels of IFNγ and / or IL-12 (advantageously, also of IFNα and / or IFNβ, and possibly of IL-4).

[0035] Preferably, in the method, the epitope has the consensus sequence X1X2X3X4X5X6X7, wherein the X2X3X4X5X6independently represent any amino acid, wherein the X1and X7residues are independently {F; W; T; H or Y}, wherein, preferably, X4is {I; V; L or M} and / or wherein, preferably, at least one of the X1or X7residues is {F; W or Y} and / or wherein, preferably, said X1X2X3X4X5X6X7sequence belongs to and / or forms an α-helix, possibly after binding to CD1.

[0036] Preferably, the method further comprises the step of (ex vivo) measuring the specific cytotoxic response to said epitope from a sample from the patient after administration of the peptide epitope.

[0037] Advantageously, in the pharmaceutical method, the peptide comprising the CD1 epitope does not comprise a class II MHC epitope.

[0038] Preferably, the pharmaceutical method further comprises a subsequent step of administering to the patient a vaccine, preferably together with the adjuvant, the vaccine being a second peptide comprising a MHC class II epitope, and preferably being a peptide further comprising a CD1 peptide epitope, and possibly comprising a MHC class I or II epitope.

[0039] Preferably, the method comprises a step of measuring the level of IgGl and / or IgG3 (from a blood sample of the patient), possibly after injection of a CD1 peptide epitope (not comprising a MHC class II epitope) and / or injection of a second peptide (comprising a CD1 peptide epitope and a MHC class II epitope), advantageously enabling the recognition of said CD1 peptide epitope. DETAILED DESCRIPTION

[0040] The inventors have found that, according to the conventional practice in the art, the vaccination of a patient with a vaccine comprising a virus, sometimes a cell-internal pathogen, is sometimes inefficient.

[0041] On the other hand, the inventors have found that, when a CD1 -restricted epitope (or a nucleotide sequence encoding it) of the same virus or cell-internal pathogen is administered prior to the vaccination procedure, the immune response to the virus or cell-internal pathogen is enhanced compared to the vaccination with (conventional) administration of a vaccine of the virus peptide or pathogen with an adjuvant: the cytotoxicity, including CD8+ T cells, NKT and NK cells, is enhanced, as well as the concentration of specific antibodies is significantly increased and the activation of CD4+ T cells. In other words, while chronic infections are mainly the result of strategies developed by the pathogen to destroy the adaptive immunity, the inventors have found that the boosting of the innate response provides the impetus for an effective adaptive response.

[0042] As a forerunner in the identification of the potential of modulating the immune system based on CD1 -peptide interactions (in the present invention, the term "CD1" refers to CDla, CDlb, CDlc and CDld, but not to CDle, unless otherwise specified), the inventors have found that the vaccination of a patient with a CD1 epitope induces or promotes the subsequent generation of a local and / or specific Thl -like response with cytotoxic function, and / or the local and / or specific production of IFNy and / or IL-12 cytokines. Another hallmark of NKT activation is the level of expression of its perforin and granzymes, which initiate the cytotoxic response.

[0043] Therefore, this response represents an added value over the conventional vaccination, since the peptide selected on a different, more targeted basis, is able to induce a more predictable response of the immune system, in this case a focused Thl -like response, instead of a less predictable one.

[0044] In fact, the present application can induce a strong immune response in a patient, dominated by cytotoxic T cells, while avoiding the deleterious effects of (i) exogenous molecules modulating immunity in a non-selective manner, and (ii) inappropriate reactions of the patient's body to intracellular pathogens.

[0045] Furthermore, the present inventors have found that such CD1-focused vaccination is synergistic with a subsequent, more traditional vaccination regimen that is not necessarily CD1 / NKT and cytotoxicity-focused, because the immune system has already been conditioned to react correctly: the reaction to the subsequent vaccination is now properly targeted and reinforced.

[0046] Thus, a first aspect of the present application is a CD1 peptide epitope for use in the prevention or treatment of a disease caused by an intracellular pathogen, wherein

[0047] (i) said disease is preferably a viral disease, caused by a virus preferably selected from the group consisting of Flaviviridae, Coronaviridae, Orthomyxoviridae, Herpesviridae and Picornaviridae,

[0048] (ii) said CD1 peptide epitope is a fragment of a protein expressed by said intracellular pathogen, wherein said CD1 peptide epitope comprises the sequence X1X2X3X4X5X6X7, wherein said X2, X3, X4, X5 and X6 independently represent any amino acid, and said X1 and X7 residues are independently {F; W; T; H or Y}, and

[0049] (iii) said use comprises administering an inactivated form of said intracellular pathogen or a second peptide based on a protein of said intracellular pathogen, wherein said inactivated intracellular pathogen or said second peptide is administered after the CD1 peptide epitope, and wherein said second peptide comprises the CD1 epitope sequence defined in (ii) and at least one MHC class I epitope.

[0050] In one embodiment, X4 is {I; V; L or M}. In another embodiment, at least one of the X1 or X7 residues is {F; W or Y}. In yet another embodiment, said X1X2X3X4X5X6X7 sequence belongs to and / or forms or has the ability to form an alpha-helix.

[0051] In one embodiment, the CD1 peptide epitope, i.e. the peptide for the first administration, does not comprise an MHC class II epitope.

[0052] In one embodiment, the CD1 peptide epitope, i.e. the peptide for the first administration, does not comprise an MHC class II epitope.

[0053] In one embodiment, said second epitope, i.e. the peptide for the second administration, further comprises at least one MHC class II epitope.

[0054] Another aspect of the application is a pharmaceutical method for modulating the immune system of a patient to produce a specific Thl-like and / or cytotoxic response against an intracellular pathogen, comprising the steps of: (in silico) identifying (putative) CD1 (CDla, CDlb, CDlc and / or CDld) peptide epitopes in said intracellular pathogen, measuring the ability of the identified (putative) CD1 (CDla, CDlb, CDlc and / or CDld) peptide epitopes to activate NKT cells in a cytotoxic response (production of IFNy and / or IL-12; expression of perforin and granzymes; measurement of3H thymidine incorporation and / or NUR77 expression) in vitro and administering said (validated) CD1 (CDla, CDlb, CDlc and / or CDld) peptide epitopes to the patient, preferably together with an adjuvant (i.e. first vaccination step, first vaccine; second vaccine see below).

[0055] It is possible that not the CD1 peptide epitopes are administered (directly), but rather nucleotide sequences encoding such CD1 peptide epitopes are administered to the patient.

[0056] Preferred pathogens are viruses (such as DNA viruses, RNA viruses (such as flaviviruses (West Nile virus, Dengue virus) or coronaviruses)), as well as bacteria and mycobacteria (such as Mycobacterium tuberculosis and other mycobacteria pathogenic to humans or animals, Yersinia, Brucella), chlamydia, mycoplasma, rickettsia, as well as parasitic protozoan diseases (e.g. Leishmania, Trypanosoma, Toxoplasma), Listeria and Histoplasma.

[0057] The in silico identification is preferably achieved after first identifying the intracellular pathogen, and then, optionally, screening suitable proteins, and then screening for a motive that is readily bound by CD1 molecules (CDla, CDlb, CDlc and / or CDld). The ability of the peptide (comprising the (putative) epitope) to bind to CD1 (CDla, CDlb, CDlc and / or CDld) molecules is best determined further in vitro.

[0058] Preferably, in this method, the NKT cell activation is after the identified (putative) peptide epitope (in silico and / or after the above-mentioned in vitro binding step) is incubated with CD1 (CDla, CDlb, CDlc and / or CDld)-bearing cells, and NKT cells are added to the population, and activation of these NKT cells is determined, e.g. by their ability to exert a cytotoxic effect on reporter cells.

[0059] Advantageously, the activation of the NKT cells is determined (also) by quantification of the level of IFNγ and / or IL-12 (quantification of the mRNA or protein level; see below) or the expression of perforin and / or granzyme and / or NUR77 level, and 3H thymidine incorporation (by the NKT cells). This ensures that the selected peptide epitope will induce the correct cytotoxic immune response when injected into the patient.

[0060] In this method (the computer steps of the method), preferably, the peptide epitope is a (CD1, e.g. CD1d) peptide epitope having the consensus sequence X1X2X3X4X5X6X7, wherein X2X3X4X5X6 independently represents any amino acid, wherein X1and X7residues are independently {F; W; T; H or Y}, wherein, preferably, X4is {I; V; L or M} and / or wherein, preferably, at least one of the X1or X7residues is {F; W or Y} and / or wherein, preferably, the X1X2X3X4X5X6X7sequence forms and / or becomes part of an alpha helix, possibly after binding to CD1.

[0061] The presence of at least one F, W or Y residue in X1or X7ensures strong anchoring on CD1.

[0062] The conformation in the helix also contributes to good affinity for CD1. This means that other amino acids, in particular at the X4position, are compatible with the alpha helix.

[0063] The presence of an alpha helix can be predicted in silico by computer. In the present invention, it is known that several amino acids that disrupt the alpha helix, such as proline or glycine, are acceptable in certain positions. For example, a glycine at residue X2, X3, X5or X6is possible, since this residue allows more flexibility, which is advantageous for the fixation of CD1. Conversely, a proline residue (for example, at the hypothetical X0or X -1、-2、-3 position) can be the first amino acid of the alpha helix. On the other hand, a large number of amino acids, such as arginine or tryptophan, are not preferred in the intermediate positions X2-X6, while tryptophan can be present, even as one of the preferred amino acids for X1and / or X7.

[0064] In addition to the in silico screening, the potential to form an alpha helix can be tested by chemical analysis such as nuclear magnetic resonance or X-ray, preferably after loading into CD1.

[0065] Thus, a preferred CD1 sequence has a W, F or Y residue in X1and / or X7, and forms (or has the ability to form) an alpha helix.

[0066] In addition, a preferred CD1 sequence has a W, F or Y residue in X1and / or X7, and an I, L, M or V residue in X4.

[0067] Most preferred CD1 sequences have a W, F or Y residue at X1and / or X7, an I, L, M or V residue at X4, and form (or have the capacity to form) an alpha helix.

[0068] In the method, alternatively or additionally, the peptide epitope (forming or having the capacity to form an alpha helix) is selected by a first computer screening step on the basis of the identification of a cluster of hydrophobic amino acids within the peptide (e.g. at least 2 hydrophobic amino acids in a range of up to 12 amino acids, up to 10, up to 7 amino acids, preferably at least 3 hydrophobic amino acids in a range of up to 12 amino acids, up to 10 or up to 7 amino acids, or at least 4 amino acids in a range of up to 12 amino acids, up to 10 or up to 7 amino acids).

[0069] The term “hydrophobic amino acid” is well understood by the person skilled in the art. In the context of the present application, it preferably refers to methionine (M), leucine (L), isoleucine (I), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0070] In the computer screening (including the computer screening of X1-X7 and the alternative computer screening), preferably, the CD1 epitope does not comprise a class II MHC epitope. Such epitopes, while desirable in traditional vaccination, are included in the subsequent vaccination steps of the present application (see below), can reduce, possibly substantially reduce, the CD1-driven response of the immune system.

[0071] One suitable way to ensure this is to select peptides that are short enough and / or to select peptides that do not have a predicted class II MHC epitope (e.g. truncated peptides that have had a putative class II MHC epitope removed). Suitable polypeptides can be selected by methods known in the art. For example, Poluektov et al., Vaccines 39: 2110-2216, 2021 describes epitope selection for SARS-CoV2.

[0072] In fact, a suitable way to avoid class II MHC epitopes is to design short peptides containing these CD1 epitopes, for example less than 50, less than 40, less than 30, less than 20 amino acids. A minimal peptide containing a CD1 epitope can have a size of 11 amino acids, including the 7-amino acid consensus described above. Thus, in one embodiment, the CD1 peptide epitope used according to the application is a fragment (i.e. a contiguous fragment) of a protein expressed by an intracellular pathogen, having a length between 11 and 50 amino acids, such as between 11 and 40 amino acids, or between 11 and 30 amino acids, or between 11 and 20 amino acids. In another embodiment, the CD1 peptide epitope used according to the application further contains flanking sequences which do not correspond to flanking regions in the protein expressed by the intracellular pathogen. Thus, in this embodiment, the CD1 peptide epitope comprises a fragment of a protein expressed by an intracellular pathogen, having a length between 11 and 50 amino acids, such as between 11 and 40 amino acids, or between 11 and 30 amino acids, or between 11 and 20 amino acids.

[0073] In fact, preferably, the CD1 epitope still forms or has the ability to form an alpha helix, which implies some biophysical constraints, including on the minimum size of the epitope, and thus also on the flanking residues: such residues do not directly participate in the fixation of CD1, but they bring conformation to the entire sequence, affecting its ability to bind to CD1.

[0074] Another way to exclude class II MHC epitopes, for example if one intends to design longer polypeptides, is to rely on computer analysis.

[0075] Another practical way is to rely on cellular tests, in which cells are transfected with class II human MHC haplotypes (several cell lines, each transfected with a different class II human MHC haplotype), then tested to check whether a peptide containing a CD1 epitope also meets the conditions of a class II MHC epitope, and to reject such a peptide. For example, 15 to 20 major MHC haplotypes can be tested.

[0076] Preferably, the method further comprises a step of measuring ex vivo a specific cytotoxic response to the CD1 epitope. This can ensure the correct activation of the immune system, in addition to the direct cytotoxic effect of the NKT cells (see below the second vaccination step), before administering the second peptide epitope to the patient.

[0077] Preferably, the method further comprises a subsequent second vaccination step, i.e. administering a vaccine to the patient, preferably based on the same protein comprising the CD1 epitope expressed by the intracellular pathogen, preferably administered together with an adjuvant. The vaccine comprises a second peptide, which second peptide comprises at least one second epitope different from the CD1 peptide epitope. Preferably, the second vaccine comprises at least one MHC class I epitope and at least one MHC class II epitope. A preferred second vaccine advantageously comprises the CD1 epitope of the first vaccine and the MHC class II epitope. Suitable MHC class II epitopes can be identified using the methods described above, i.e. the in silico prediction methods and / or tests performed on cell lines expressing human MHC haplotypes of class II. Suitable MHC class I epitopes can be identified by methods known in the art, such as the methods described in Yang et al. in BMC Bioinformatics 22:231, 2021.

[0078] The second vaccination step can be performed after ensuring that the NKT cells have been properly activated by the first (CD1-restricted) vaccine to induce a cytotoxic response.

[0079] The epitope(s) or peptide(s) used for the second vaccination step do not necessarily focus on the CD1 epitope: peptides with multiple epitopes and peptides with MHC class II epitopes can be used, and can advantageously be used. The peptide(s) used in the subsequent vaccination can also comprise (putative) CD1 epitopes, even verified CD1 epitopes, preferably the CD1 epitope used in the first vaccination.

[0080] In some embodiments, the second step comprises administering a full-length protein of the intracellular pathogen, preferably a protein that also comprises the CD1 peptide epitope, or an inactivated form of the pathogen, such as an attenuated live virus.

[0081] In one embodiment, the pathogen is Dengue virus, and the CD1 peptide epitope is a fragment of the NS protein, preferably NS5 and comprising or consisting of the sequence listed in SEQ ID NO: 1. Preferably, the CD1 peptide epitope has a length between 11 and 50 amino acids, such as between 11 and 40 amino acids, or between 11 and 30 amino acids, or between 11 and 20 amino acids. In yet another embodiment of the application, the second peptide is the full-length NS5 protein.

[0082] In another embodiment, the pathogen is Coxsackievirus B4, and the CD1 peptide epitope is a fragment of the polyprotein, preferably comprising or consisting of the sequence listed in SEQ ID NO: 2. Preferably, the CD1 peptide epitope has a length between 11 and 50 amino acids, such as between 11 and 40 amino acids, or between 11 and 30 amino acids, or between 11 and 20 amino acids. In yet another embodiment of the application, the second peptide is the full-length polyprotein.

[0083] In another embodiment, the pathogen is SARS-CoV2 virus and the CD1 peptide epitope is a fragment of the S protein, preferably comprising or consisting of the sequence listed in SEQ ID NO: 3. Preferably, the CD1 peptide epitope is between 11 and 50 amino acids in length, such as between 11 and 40 amino acids, or 11 and 30 amino acids, or 11 and 20 amino acids. In yet another embodiment of the application, the second peptide is the full-length S protein.

[0084] In another embodiment, the pathogen is Influenza A virus and the CD1 peptide epitope is a fragment of the hemagglutinin protein, preferably comprising or consisting of the sequence listed in SEQ ID NO: 4. Preferably, the CD1 peptide epitope is between 11 and 50 amino acids in length, such as between 11 and 40 amino acids, or 11 and 30 amino acids, or 11 and 20 amino acids. In yet another embodiment of the application, the second peptide is the full-length hemagglutinin.

[0085] In another embodiment, the pathogen is Herpes simplex virus and the CD1 peptide epitope is a fragment of the Vpl6 protein, preferably comprising or consisting of the sequence listed in SEQ ID NO: 5. Preferably, the CD1 peptide epitope is between 11 and 50 amino acids in length, such as between 11 and 40 amino acids, or 11 and 30 amino acids, or 11 and 20 amino acids. In yet another embodiment of the application, the second peptide is the full-length Vpl6 protein.

[0086] A related aspect of the application is an in vitro method for identifying a peptide epitope to activate NKT cells to induce (subsequent) cytotoxic responses (IFNy and / or IL-12; levels of perforin, granzyme and / or NUR77 expression; 3H thymidine incorporation) against an intracellular pathogen, the in vitro method comprising the steps of identifying a CD1 (CDla, CDlb, CDlc and / or CDld) epitope in the intracellular pathogen and measuring in vitro the ability of the identified CD1 (CDla, CDlb, CDlc and / or CDld) epitope to activate NKT cells in a cytotoxic response.

[0087] Preferably, in the in vitro method, the CD1 epitope (CDla, CDlb, CDlc and / or CDld epitope(s)) is identified on the basis of the ability of the peptide to bind the CD1 (CDla, CDlb, CDlc and / or CDld) molecule.

[0088] Preferably (or in addition to the binding to CD1), in the preliminary (in silico) step in the pharmaceutical method as described above, peptides are identified that can bind to the CD1 (CD1a, CD1b, CD1c and / or CD1d molecule(s)) (in the presence of a stretch of hydrophobic amino acids or in the presence of an X1X2X3X4X5X6X7epitope, with specific amino acids at positions 1, 7 (F / W / T / H / Y) and possibly also at position 4 (I / L / M / V)).

[0089] Preferably, or in addition, in the in vitro method, the activation of NKT cells is measured by incubating the peptide epitope with CD1 (CD1a, CD1b, CD1c and / or CD1d)-bearing cells, followed by the addition of a population of NKT cells and determining the activation of these NKT cells.

[0090] Preferably, in the method (possibly also for the pharmaceutical method described above or the pharmaceutical product below), the activation of NKT cells is determined by quantifying the level of IFNy and / or IL-12, for example based on the expression level of mRNA for IFNy and / or IL-12 or the amount of protein in the surrounding (modulated) cell culture medium, for example measured by ELISA. It can also be advantageous to measure the expression level of perforin and granzymes, as well as NUR77 (mRNA level) or 3H thymidine incorporation (in such NKT cells).

[0091] A related aspect of the application is a CD1 (CD1a, CD1b, CD1c and / or CD1d) peptide epitope (vaccine) for use in the treatment of a disease caused by an intracellular pathogen (preferably a virus).

[0092] Alternatively, instead of administering a CD1 peptide epitope directly, the corresponding nucleotide sequence expressing the CD1 peptide epitope can be administered to the patient.

[0093] Although not really limited to a particular viral disease or virus, the viral disease is preferably selected from the group consisting of flaviviruses (West Nile virus, Dengue virus), coronaviruses (SARS, such as SARS-CoV-2), orthomyxoviruses (viruses causing influenza), herpesviruses (herpesviruses, including Epstein-Barr virus (EBV)) and picornaviruses. In the context of the present application, a viral disease also includes malignancies associated with viruses, such as lymphomas associated with EBV, in particular EBV-associated lymphomas occurring after organ transplantation, such as liver transplantation.

[0094] In addition, intracellular infections with bacteria and mycobacteria (e.g. Mycobacterium tuberculosis and other mycobacteria pathogenic for humans or animals (Yersinia, Brucella), chlamydia, mycoplasma, rickettsia and parasitic protozoan diseases (e.g. Leishmania, trypanosomes, Toxoplasma), Listeria and Histoplasma can be treated by CD1 peptide vaccination according to the present application.

[0095] Preferably, the CD1 (CD1a, CD1b, CD1c and / or CD1d) peptide epitope is obtained according to the in vitro method described above and / or according to the same method as described above for the identification of a peptide epitope for the activation of NKT cells, in particular comprising specific computer step(s), in vitro (CD1 binding) step(s) and / or step(s) for the (in vitro) measurement of NKT cell activation.

[0096] A further related aspect of the present application is a kit for the treatment of a disease caused by an intracellular pathogen, the kit comprising a CD1 (CD1a, CD1b, CD1c and / or CD1d) peptide epitope (preferably a CD1a, CD1b, CD1c, and / or CD1d peptide epitope as described above, and not comprising a class II MHC epitope) and a second peptide, preferably comprising a class II MHC epitope derived from the intracellular pathogen, preferably belonging to the same protein as the CD1 peptide epitope, more preferably the second peptide comprises at least the CD1 peptide epitope and class II MHC epitope(s).

[0097] The kit is advantageously used for the treatment of a disease caused by an intracellular pathogen (e.g. a virus, also including intracellular bacteria) (see above).

[0098] Preferably, the kit further comprises an adjuvant for the administration of the CD1 (CD1a, CD1b, CD1c and / or CD1d) peptide epitope for vaccination and / or an adjuvant for the administration of the second peptide for vaccination.

[0099] The preferred adjuvant is selected for its ability to induce an inflammatory response, such as a lipid emulsion. Alum adjuvants are not preferably used for CD1 peptide epitopes.

[0100] Alternatively, instead of the CD1 peptide epitope of the present kit, the corresponding nucleotide sequence expressing the CD1 peptide epitope can be administered to the patient.

[0101] Examples

[0102] 1. Dengue virus infection

[0103] Dengue is a major public health problem in the tropics and sub-tropics. This ssRNA enveloped flavivirus is responsible for a febrile syndrome, the manifestations of which are severe hemorrhages, which can lead to shock syndrome. Despite the fact that the virus is activating the interferon a / b response, this interferon a / b response is severely impaired by many mechanisms which hamper an effective response. Inducing a strong innate response to the dengue virus would help to control the infection by its ability to elicit a CD8+ T cell response known to be protective, while the exact role of antibodies remains controversial.

[0104] Given the importance of the immune response to NS for the production of an effective viral protection, the non-structural (NS) proteins were used to develop a vaccination strategy. Thus, according to the importance of NS5 in blocking the signal required for the production of interferon a and b, NS5 was chosen for the following experiments.

[0105] The sequence of NS5 shows determinants with the characteristics required for binding to CD1, namely

[0106] AG- HGQVDNF -SL (SEQ ID NO: 1), corresponding to amino acids 1127 to 1137 with the motif underlined.

[0107] The peptide of SEQ ID NO: 1 was emulsified in complete Freund's adjuvant and injected subcutaneously into a series of C57BL / 6 mice at a dose of 100 pg, 2 weeks later, the peptide was injected at the same dose in incomplete Freund's adjuvant. The control group was injected with the adjuvant alone according to the same timetable.

[0108] Two weeks after the last injection, the two groups of mice were treated by subcutaneous injection of NS5 protein in alum, repeated four times every 2 weeks.

[0109] The spleens of all the mice were taken one month after the last injection and the CD3+ lymphocyte population was isolated by FACS sorting.

[0110] The cells were then maintained in culture in the presence of antigen-presenting cells loaded with NS5, in proportion to the activated cells counted by 3H-thymidine incorporation 6 days later.

[0111] The results show that the total number of NS5-specific cells is significantly higher in mice that received the CD1 -restricted peptide before vaccination. This higher number is observed for CD4, and most importantly, this higher number is very significant for CD8 cells.

[0112] In parallel experiments, CD3+ cells were incubated with JAWS2 as presenting cells. This cell line expresses high levels of surface CD1 but not class II MHC complexes. NS5-specific NKT cells were detected by staining with PLZF antibody and were found only in the group of mice pre-sensitized with the peptide of SEQ ID NO: 1.

[0113] It can thus be concluded that the pre-injection of CD1 -restricted epitopes in the adjuvant significantly enhances the overall immune response to NS5 but is particularly efficient in enhancing the cytotoxic immune response characterized by class I -restricted CD8+ T cells and NKT cells.

[0114] Since C57BL / 6 and BALB / c mice differ in the frequency and distribution of NKT cells, the same experiment was repeated in BALB / c mice with essentially the same results.

[0115] It is worth noting that the number of CD4, CD8 and NKT cells varied little when comparing individuals in the group previously injected with CD1 -restricted epitopes or individuals in C57BL / 6 and BALB / c mice, whereas greater variations were observed between individual mice in the control group of either strain.

[0116] It can be concluded that the pre-immunization with CD1 -restricted peptides not only increases the overall immune response to NS5 but also reduces the differences usually observed when comparing mice of different strains.

[0117] 2. Coxsackie virus B4 infection

[0118] Coxsackie virus B4 is a ssRNA picornavirus associated with myocarditis and pancreatitis. Intraperitoneal injection of certain coxsackie virus strains, such as B4-V, in mice immediately triggers acute pancreatitis, followed by a chronic inflammation phase. In the BALB / c strain, the transition from acute to chronic inflammation relies on IL-10, a typical type 2 cytokine, and the absence of IL-10 leads to an interrupted form of acute inflammation and failure to enter the chronic phase.

[0119] Although coxsackie virus B4-V activates innate immunity, including TLR3, TLR4 and TLR7, the virus manages to circumvent this innate response and replicates in the pancreatic acinar lobules in the context of a Th2 immune response. Enhancing the immune response to a cytotoxic response would prevent infection and / or at least prevent the transition from acute to chronic pancreatitis.

[0120] The amino acid sequence of the coxsackie virus B4 polyprotein contains hydrophobic epitopes

[0121] EK- FRDIRGF- LA (SEQ ID NO: 2), corresponding to amino acids 1619 to 1629 of the CD1 binding motif with underlined

[0122] BALB / c mice were immunized by subcutaneous injection of 100 pg of the peptide of SEQ ID NO: 2 emulsified in Freund’s adjuvant, once every 2 weeks for a total of two times. The control group was injected with adjuvant alone.

[0123] All mice were then immunized with whole multimer adsorbed on alum, 100 pg per injection, administered every 2 weeks for a total of 4 injections.

[0124] Three weeks after the last injection, all mice were infected in the peritoneum with 50 PFU of the B4-V strain.

[0125] Within 3 days of infection, the pancreas of 50% of the mice was isolated and examined for cellular infiltration. All mice in the control group showed massive infiltration of mononuclear cells in the acini. Mice treated by injection of the peptide SEQ ID NO: 2 showed a moderate infiltration of such cells.

[0126] However, on day 10 post-infection, all remaining mice in the control group showed persistent cellular infiltration and loss of acinar structure, while mice pre-treated with the peptide of SEQ ID NO: 2 showed complete resolution of the pancreatitis.

[0127] It can therefore be concluded that pre-immunization with the peptide of SEQ ID NO: 2 reduces the extent of acute acinar inflammation due to B4-V infection and, more importantly, prevents the pancreatitis from entering the chronic irreversible phase.

[0128] 3. SARS-CoV-2 infection

[0129] SARS-CoV-2 is the causative agent of the coronavirus disease (2019 and onwards) (CoVid-19) pandemic. Observations on the spontaneous immune response in patients suggest that a severe dysregulation of the immune response and inflammation is associated with adverse outcomes. SARS-CoV-2 has developed several strategies to evade innate immunity, leading to insufficient production of interferon a / b and a preferential Th2 adaptive response, and the risk of immunopathology and antibody-dependent enhancement remains to be addressed.

[0130] Redirecting the response to effective innate immunity not only provides significant protection but also prevents secondary complications.

[0131] SARS-CoV-2 cell invasion relies on the interaction between the viral spike protein (S protein) and the angiotensin 2 receptor (ACE2). Therefore, the development of an immune response to the S protein would constitute an appropriate target for infection control.

[0132] To establish an efficient S-protein specific innate response, a peptide comprising amino acid residues organized in a motif suitable for CD1 presentation was identified using the algorithm described in patent EP3388447 Al.

[0133] Thus, the sequence ATR FASVYAW NRK (SEQ ID NO: 3) contains such a motif (underlined), corresponding to residues 353 to 365 of the SARS-CoV-2 spike protein.

[0134] C57BL / 6 mice were immunized with the peptide of SEQ ID NO: 3 emulsified in complete Freund’s adjuvant at a dose of 100 pg, then 3 weeks later, a second injection of 100 pg of the peptide emulsified in incomplete Freund’s adjuvant. The control group received only the adjuvant.

[0135] Both groups of mice were then immunized with 100 pg of S-protein in alum, administered 2 weeks apart, starting 3 weeks after the injection of the peptide of SEQ ID NO: 3 (or adjuvant), for a total of 4 injections.

[0136] The immune response to S-protein was then evaluated on splenocytes retrieved 2 or 6 months after the last injection, and the titer of specific antibodies was evaluated on sera.

[0137] The results show that mice pre-treated with the peptide of SEQ ID NO: 3 have a significantly higher number of CD3+ lymphocytes to the protein S compared to control mice. Moreover, the results show that at 2 months, the number of CD8+ T cells is greater than the number of CD4+ T cells in the pre-treated group, while the opposite is true for the control group. Notably, the same trend towards preferential CD8+ T cells is also observed 6 months later.

[0138] The evaluation of specific anti-S-protein antibody titers at 2 months and 6 months after the end of the immunization protocol shows that mice pre-treated with the peptide of SEQ ID NO: 3 have significantly higher titers at both time points. The isotype distribution of the antibodies shows a dramatic shift from IgGl to IgG2a in the control group to the group pre-treated with the peptide of SEQ ID NO: 3, and this shift is maintained 6 months later.

[0139] It can thus be concluded that pre-treating mice with a peptide comprising a CD1 compatible motif increases the immune response to S-protein, changes its properties towards increased cytotoxicity and Thl driven response, and maintains this immune response over time.

[0140] 4. Influenza virus infection

[0141] Influenza virus infection has a major impact worldwide and causes significant mortality. One of the obstacles that hinders the success of vaccination strategies is the high mutation rate of the virus, which forces the design of new vaccines every year. In addition, the clinical and immunological outcome of exposure to the virus depends on the individual's past history: vaccination and / or natural exposure to the virus reactivates the response to the previous virus, a phenomenon known as "antigenic priming". This phenomenon makes it unlikely that the current vaccination strategy will develop a "universal" vaccine, although certain determinants of the virus are more conserved than others and can be used for such a vaccination.

[0142] Creating an immunological environment that is easy to induce a response to the virus of the innate immunity would be an important step forward for a universal vaccine, and has the property of replacing the nature and quality of the response made previously to the vaccine and / or exposure to the virus.

[0143] Thus, the hemagglutinin from influenza A virus contains a sequence corresponding to residues 345 to 355: GL- FGAIAGF - IE (SEQ ID NO: 4), which shows a consensus motif (underlined) capable of activating NKT cells.

[0144] C57BL / 6 mice in a small path were immunized with a synthetic peptide comprising SEQ ID NO: 4 emulsified in Freund's adjuvant at a dose of 100 μg. This injection was repeated 2 weeks later, but using incomplete Freund's adjuvant.

[0145] The control mice were injected using a similar protocol, but with the adjuvant only.

[0146] Three weeks after the last injection, all the mice were injected with 100 μg of hemagglutinin adsorbed on alum, and the injection was repeated 3 times at two-week intervals.

[0147] One month after the last injection, all the mice were sacrificed and lymphocyte populations were prepared from the spleen and the local lymph nodes.

[0148] Significant quantitative and qualitative differences were observed between the two groups of mice. The response of the mice immunized with the peptide of SEQ ID NO: 4 was mainly Thl-like and cytotoxic to hemagglutinin (including CD8+, NK and NKT cells), as identified with lineage-specific markers, while the control mice essentially showed a Th2-type immune response, with little cytotoxic involvement. Thus, when the bulk lymphocyte populations were incubated in the presence of histocompatibility antigen-presenting cells loaded with hemagglutinin, the cytokine production by the lymphocytes of the immunized mice showed a higher ratio of IFN-γ to IL-4, while the control mice showed the opposite.

[0149] The isotype profile of the anti-hemagglutinin antibodies in the serum showed that in the mice immunized with the peptide of SEQ ID NO: 4, the concentration of IgG2a was very high, while IgGl dominated the response produced in the control mice, which is consistent with the domination of Thl-like over Th2 response, respectively.

[0150] Thus, it can be concluded that the pre-immunization with the peptide activating NKT cells changed the quality of the immune response obtained after the administration of hemagglutinin, towards a Thl -driven response and cytotoxicity.

[0151] 5. Herpes simplex virus infection

[0152] Type 1 or 2 alpha herpes simplex virus (HSV) are ubiquitous pathogens that persist throughout the life of the infected individual. They constitute a latent viral pool in the terminally differentiated neurons of the peripheral ganglia. The response of the immunocompetent individual results in a self-limiting illness that parallels a strong Thl-like and cytotoxic immune response. However, this does not exclude severe symptoms and significant morbidity, especially in immunocompromised hosts. When the virus enters the lytic phase, viral particles are shed and taken up by antigen presenting cells within the ganglia. The strength of the T-dependent cytotoxic activity will determine the degree to which the infection can be controlled.

[0153] Interestingly, HSV has already elucidated a strategy to prevent CD1d viral presentation, pointing to the importance of this pathway in defense against viruses. Moreover, additional mechanisms to avoid NK cell clearance have been identified and the accumulation of Thl cells at the site of infection is directly proportional to the control of this infection. In summary, forcing a viral response by CD1 and its impact on NK cell activation would be an important step in the control of HSV infection.

[0154] Vaccination with live attenuated viruses currently induces B and T lymphocytes, but its overall activity in the elderly is at most 50%, not to mention the risk of inducing a latent infection. Thus, there is an important unmet medical need in terms of HSV vaccination strategies.

[0155] The epitope of the HSV Vpl6 transcriptional regulatory protein comprises the sequence VL-FLHLYLF-LT (SEQ ID NO: 5) corresponding to amino acid residues 179-189, and the CD1 binding motif is underlined.

[0156] BALB / c mice in the path were immunized with 100 μg of the peptide of SEQ ID NO: 5 emulsified in complete Freund's adjuvant and the injection was repeated once, 2 weeks later, the second injection being performed in incomplete Freund's adjuvant. The control group of BALB / c mice was treated with adjuvant alone.

[0157] Then, all the mice received two times, one month apart, a commercial live attenuated HSV preparation.

[0158] One month after the last injection, the immune responses to the vaccine were compared between the two groups. The results showed that pre-immunization with the peptide of SEQ ID NO: 5 shifted the response to a Thl -like environment, including a significant expansion of cells sharing cytotoxic potential, CD8+ T cells, NK cells and NKT cells. The concentration of total anti-HSV antibodies was similar in both groups, but the pre-immunized mice showed a significant increase in the production of IgG2a and IgG2b specific antibodies, as shown by direct binding ELISA.

[0159] Thus, it can be concluded that immunization with a peptide containing a CD1 binding motif is sufficient to shift the response to acquire cytotoxic properties that help control HSV infection. SEQUENCE LISTING <110> Ikivo Limited <120> Tuning of the immune system <130> PAT84208EP00 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CD1 NS Dengue virus <220> <221> MISC_FEATURE <222> (3)..(9) <223> CD1 epitope <400> 1 Ala Gly His Gly Gin Val Asp Asn Phe Ser Leu 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Coxsackievirus B4 CD1 epitope <220> <221> MISC_FEATURE <222> (3)..(9) <223> CD1 core epitope <400> 2 Glu Lys Phe Arg Asp lie Arg Gly Phe Leu Ala 1 5 10 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> SARS COVID Spike Protein CD1 Epitope <220> <221> MISC_FEATURE <222> (4)..(10) <223> CD1 Core Epitope <400> 3 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Influenza Virus Hemagglutinin Protein CD1 Epitope <220> <221> MISC_FEATURE <222> (3)..(9) <223> CD1 Core Epitope <400> 4 Gly Leu Phe Gly Ala lie Ala Gly Phe lie Glu 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HSV Vpl6 CD1 Epitope <220> <221> MISC_FEATURE <222> (3)..(9) Core CD1 epitopes <400> 5 Val Leu Phe Leu His Leu Tyr Leu Phe Leu Thr 1 5 10

Claims

1. Use of a first peptide comprising a CD1 peptide epitope and a second peptide for the manufacture of a medicament for the prevention or treatment of a disease caused by an intracellular pathogen, wherein (i) the disease is a viral disease caused by a virus selected from the group consisting of a flavivirus, a coronavirus, an orthomyxovirus, a herpesvirus and a picornavirus, (ii) the CD1 peptide epitope is a fragment of a protein expressed by the intracellular pathogen, wherein the CD1 peptide epitope comprises the sequence X1X2X3X4X5X6X7, wherein X2, X3, X4, X5 and X6 independently represent any amino acid, wherein the X1 and X7 residues independently are {F; W; T; H or Y}, and (iii) the use comprises administration of an inactivated form of the intracellular pathogen or administration of a second peptide based on a protein of the intracellular pathogen, wherein the administration of the inactivated intracellular pathogen or the second peptide is performed after administration of the CD1 peptide epitope, and wherein the second peptide comprises the CD1 epitope defined in (ii) and at least one MHC class I epitope.

2. Use according to claim 1, wherein X4 is {I; V; L or M} and / or wherein at least one of the X1 or X7 residues is {F; W or Y} and / or wherein the X1X2X3X4X5X6X7 sequence forms or has the capacity to form an alpha helix.

3. Use according to claim 1, wherein the CD1 peptide epitope does not comprise an MHC class I epitope.

4. Use according to the preceding claim 1, wherein the CD1 peptide epitope does not comprise an MHC class II epitope.

5. Use according to the preceding claim 1, wherein the second peptide further comprises at least one MHC class II epitope.

6. Use according to the preceding claim 1, the CD1 peptide epitope is obtainable by an in vitro method for identifying one or several peptide epitopes to activate NKT cells and / or cytotoxic responses in Th1-like against an intracellular pathogen, said in vitro method comprising the steps of: - identifying CD1 peptide epitopes in the intracellular pathogen, and - measuring in vitro the capacity of the identified CD1 peptide epitopes to activate NKT cells and / or cytotoxic responses in Th1-like. In the in vitro method, the activation of NKT cells is measured after incubation of the peptide epitopes with a population of NKT cells following incubation of the peptide epitopes with cells expressing CD1 molecules on their surface and determination of the activation of the NKT cells. In the in vitro method, the identification of CD1 peptide epitopes is measured according to the capacity of peptides of the intracellular pathogen to bind to CD1 molecules. In the in vitro method, the activation of NKT cells is determined by quantification of interferon gamma and / or interleukin-12 levels.

10. A kit for the treatment of a disease caused by an intracellular pathogen, comprising a first peptide which is the first peptide comprising a CD1 peptide epitope according to the use of any one of the preceding claims 1 to 9, and a second peptide derived from the intracellular pathogen and further comprising a CD1 peptide epitope. ​ 7. Use according to claim 6, wherein, ​ 8. The use according to claim 6, wherein, ​ 9. Use according to any one of the preceding claims 6 to 8, wherein, ​ ​ 11. The kit according to claim 10, wherein the second peptide further comprises at least one MHC class II epitope.

12. The kit according to claim 10, for use in the treatment of a viral disease, said disease being caused by a virus selected from the group consisting of Flaviviridae, Coronaviridae, Orthomyxoviridae, Herpesviridae and Picornaviridae.

13. The kit according to claim 10, further comprising an adjuvant for the first peptide vaccination and / or an adjuvant for the second peptide vaccination.

14. The kit according to the preceding claim 10, further comprising means for quantifying IgG2 specific for the CD1 peptide epitope and / or means for quantifying IgGl and / or IgG3 specific for the CD1 peptide epitope.

15. The kit according to any one of the preceding claims 10 to 14, further comprising means for quantifying IL-12, IFNy, IL-4, IFNa and / or IFNp.