Ex vivo human model intended for evaluating the vaccine potential of a composition

Abstract

The present invention relates to an in vitro method for determining the vaccine potential of a composition comprising the steps of: ia) transcutaneous administration of the composition to a skin explant, comprising the epidermis, dermis and epidermal appendages as well as a thickness of at least 5 millimeters of hypodermis; ib) the activation status of the antigen-presenting cells within the skin explant; etii) determination of the vaccine potential of the composition.

Description

Title of the invention: Ex vivo human model for evaluating the vaccine potential of a composition technical field

[0001] The present invention relates to the field of vaccination and proposes, more specifically, the first ex vivo human model allowing the determination of the vaccine potential of a composition. Previous art

[0002] To ensure its protection, the human body has 2 types of defense mechanisms which are innate immunity and adaptive immunity.

[0003] Innate immunity allows the body to defend itself against infectious agents immediately. Conversely, adaptive (or acquired) immunity provides the body with protection that is delayed but more lasting.

[0004] In relation to innate immunity, this response is immediate in the event of exposure to an infectious agent. Furthermore, this response is independent of the antigens of said infectious agent. Moreover, this immune response is of comparable intensity with each exposure to the same infectious agent.

[0005] Adaptive immunity requires a learning phase for the organism. Following the interaction between an infectious agent and innate immunity, adaptive immunity becomes active in lymphoid tissues, especially in the lymph nodes and spleen. Several mechanisms then come into play:

[0006] 1- The antigen (infectious agent) directly activates B lymphocytes, which They possess specific receptors. Activated B lymphocytes then become plasma cells, which will secrete specific antibodies for the destruction of the antigen (humoral immunity).

[0007] 2 - The antigen (infectious agent) is presented to T lymphocytes by cells Antigen-presenting cells (e.g., dendritic cells). Antigen-presenting cells activate T lymphocytes, which differentiate into: • Cytotoxic T lymphocytes (CD8+), which destroy infected cells (cellular immunity); • Helper T lymphocytes (CD4+), or T follicular helper cells, which stimulate B lymphocytes to produce a greater quantity of antibodies and memory cells, which will then go to reside in the bone marrow.

[0008] Adaptive immunity therefore requires, after initial contact with an infectious agent, at least 2 to 3 weeks to develop. This immunity is dependent and specific to the antigens of an infectious agent. The immune response associated with it increases in intensity following each contact with the same infectious agent.

[0009] The vaccination strategy aims to develop adaptive immunity using antigens from a specific infectious agent. However, obtaining a composition with good vaccine potential is a complex undertaking. First and foremost, it is essential that this composition allows for the proper activation of antigen-presenting cells, so as to induce a specific immune response. Simultaneously, it is desirable that this composition not induce an excessively strong inflammatory response (i.e., limited mast cell degranulation).

[0010] Today, the only option for estimating the vaccine potential of a composition lies in animal models which nevertheless leave many unknowns about the real vaccine potential of a given composition in humans.

[0011] Also, there is a need for a tool that would make it possible to determine easily and quickly, and without resorting to animals, the vaccine potential in humans of an injectable composition. Description of the invention

[0012] The inventors have previously developed an ex vivo model of human skin which allows testing the subcutaneous injection of a solution without resorting to animals.

[0013] The inventors had also demonstrated that not only were mast cells stably present in this human skin model, but also that they remained functional with an intact degranulation capacity.

[0014] Now, the inventors have shown that antigen-presenting cells are also present in this skin model and furthermore that they exhibited an intact maturation capacity. This result was all the more unexpected since, following the culturing of human skin, the presenting cells were described as migrating out of the skin expiry into the culture medium (LENZ et al., J. Clin. Invest., vol.92, p:2587-2596, 1993; POPE et al., J. Invest. Dermatol., vol,104(l), p:11-17, 1995; RATZINGER et al., J. Immunol., vol.168, p:4361-4371; 2002; KIVINEN et al., Experimental Dermatology, vol. 12, p:53-60, 2003).

[0015] In view of this discovery, the inventors had the idea of ​​orienting this model so as to determine the vaccine potential of a composition by following the response of these antigen-presenting cells following the injection of this composition.

[0016] The inventors finally demonstrated that this new model effectively makes it possible to determine the vaccine potential of an ex vivo composition, which constitutes a major advance for this technological field.

[0017] Also, a first object of the invention relates to an in vitro process for determining the vaccine potential of a composition comprising the steps of:

[0018] ia) transcutaneous administration of the composition to a skin expiant comprising the epidermis, dermis and epidermal annexes as well as a thickness of at least 5 millimeters of hypodermis;

[0019] ib) determination of the activation status of antigen-presenting cells within the skin expiry; and

[0020] ii) determination of the vaccine potential of the composition.

[0021] According to a preferred embodiment, the method further includes a step ic) of determining the possible migration of antigen-presenting cells within the skin expiry.

[0022] According to another preferred embodiment, the process further comprises a step id) of determining the expression profile of genes related to the immune system, in particular cytokines, within the skin expiry and in that step ii) further allows the determination of the inflammatory potential associated with this same composition.

[0023] According to a last preferred embodiment, the process further includes a step ie) of determining the level of mast cell degranulation within the skin expiry and in that step ii) also makes it possible to determine the inflammatory potential associated with this same composition. Description of the drawings

[0024] [Fig.1] represents the different populations of antigen-presenting cells (APCs) and the markers associated with each of them. Detailed description of the invention

[0025] By “vaccine composition”, we mean a composition comprising at least one antigen (lipid, carbohydrate, protein or peptidic) or a nucleic acid encoding at least one antigen and, potentially, at least one adjuvant.

[0026] By "skin expiant" is meant a fragment of skin which includes, in addition to the epidermis, the dermis and the epidermal annexes, a thickness of at least 5 mm of hypodermis (preferably between 5 and 15 mm of hypodermis and, even more preferably, between 5 and 10 mm of hypodermis).

[0027] Epidermal appendages correspond to hair follicles, sebaceous glands, and sweat glands. The hypodermis is the layer of tissue located immediately beneath the dermis of the skin. The hypodermis is a loose connective tissue that is richly vascularized and also contains adipose tissue and immune cells.

[0028] If this skin expiant is taken from a mammal, either human or pig may be used. However, given the preferred application of the process according to the invention, a human skin expiant will be chosen instead.

[0029] In connection with the origin of the skin expiant, this can come from a plasty from any part of the body, including plasties of the abdomen, breasts, buttocks, back, or even, why not, the scalp or any other part of the body comprising skin.

[0030] The skin explant is prepared as described in international application WO 2019 / 170281.

[0031] In detail, this skin expiant was included, with the exception of the epidermis, in a liquid matrix capable of solidifying such as blood plasma, a blood plasma-derived solution (e.g., a blood plasma dilution in physiological buffer, in particular a blood plasma dilution of at least 10%, 20%, 30%, or even at least 40% (weight / total weight of the matrix)), a fibrinogen solution, a collagen solution, a gelatin solution, synthetic polymer solutions, natural polymer solutions (e.g., agarose (low / low melting point agarose or agar-agar), starch, polysaccharides), and mixtures thereof. For more details relating to the matrices capable of solidifying and the methods for placing the skin expiants in them, European patent no. EP 2 882 290 Bl. Now, these matrices and methods are described in paragraphs

[0024] to

[0042] and

[0067] to

[0080] of European patent no. EP 2 882 290 Bl.

[0032] In the case of a cylindrical skin expansive, we will therefore opt for a skin expansive with a diameter between 10 mm and 50 mm, preferably between 15 mm and 40 mm.

[0033] Advantageously, the skin expiratory material is positioned within an insert that can take various forms, including a suspended insert or an insert on stilts. A suspended insert is preferred. The base of this insert consists of a porous membrane with a diameter between 5 and 40 mm, and more preferably between 9.5 and 30 mm. The porosity of this membrane must prevent the liquid matrix from passing through it before solidification. Typically, this porous membrane will have a porosity between 0.4 and 8 µm, preferably between 0.4 µm and 1.5 µm, with the range of 0.8 µm to 1.2 µm being the preferred porosity range. As for the material, a porous membrane made of polyethylene terephthalate (PET), nitrocellulose, or polycarbonate may be chosen.Finally, and as an example of such inserts, we can cite those supplied by the companies NUNC, CORNING, BECTON DICKINSON (BD FALCON), MILLIPORE (MILLICELL), which can take the form of inserts with a membrane. made of polycarbonate, PET or nitrocellulose, which are pre-packed in multi-well plates for 6, 8, 12 or 24 well culture plates, and whose membrane porosity can vary from 0.4 and 8pm.

[0034] Transcutaneous administration refers to the administration of a composition that allows it to penetrate the skin barrier. Such transcutaneous administration can be carried out subcutaneously (i.e., by subcutaneous injection (a needle), transdermally (via a simple patch or one with microneedles) or topically.

[0035] By "topical administration" is meant an application of the composition to be tested onto the epidermis of the skin expiry in the form of a cream or a gel, for example.

[0036] By "subcutaneous administration" is meant a subcutaneous injection, which is therefore carried out in the hypodermis of the skin expiry using a needle, which is why it is also called a "hypodermic" injection. This type of injection, which is well known to those skilled in the art, generally requires creating a skin fold with the fingers, and the subcutaneous injection is then carried out in the skin fold.

[0037] By "transdermal administration", we also speak of "transepidermal administration", we mean an administration which uses a patch incorporating or not microneedles (which microneedles may be biodegradable).

[0038] According to a preferred embodiment, step ia) consists of subcutaneous or transdermal administration of a composition comprising the substance in a skin expiant.

[0039] The composition in question is a test composition which is in liquid form. Advantageously, the volume of this composition is between 10 lp and 1 ml, preferably between 10 lp and 500 lp and, particularly preferably, between 10 lp and 200 lp.

[0040] The needle for injecting the composition typically has sufficient length to reach the hypodermis. Therefore, needles with a length of 10 mm or more are preferably used. Examples of such needles include those with lengths of 12, 16, 20, 25, 30, 35, 40, or 45 mm. Advantageously, the needle has a length between 16 and 45 mm, preferably between 20 and 40 mm. The diameter of the needle to be used can be easily determined by a person skilled in the art, based on their general knowledge. Typically, such hypodermic needles are of the 18G, 19G, 20G, 21G, 22G, 23G, 25G, 26G, 27G, 28G, 29G, 30G or even 31G type.

[0041] This injection step can be performed by an experimenter, who applies pressure to the skin to allow the formation of a skin fold and thus facilitate Subcutaneous injection. Now, this injection step can also be performed using an automated injection device. Typically, the device allows for injection at a predetermined depth relative to the surface of the epidermis, thus achieving a subcutaneous injection.

[0042] Advantageously, only one step ia) of administration of a composition is carried out by skin expiant.

[0043] Step ib) of determining the activation status of immune cells and in particular of antigen-presenting cells within the skin expiry is carried out by monitoring the expression of activation markers within these cells.

[0044] To achieve this, well-known immunohistochemical methods can be used, which involve fixing the skin sample, embedding the sample (e.g., paraffin, OCT, EPON) before preservation, and finally preparing histological sections of the embedding block. Details of such methods are described, for example, in "Immunohistochemistry: Basics and Methods" by Igor Buchwalow (Springer Publishers).

[0045] Methods such as flow cytometry and BULK and SINGLE-CELL type transcriptomic analyses may also be used.

[0046] The usable histological sections may have a very significant thickness, up to 500 µm. Advantageously, the histological section will thus have a thickness between 1 and 500 µm. However, it is now possible to use sections with more conventional dimensions and a thickness between 2 and 25 µm.

[0047] Antigen-presenting cells are defined as cells expressing the CD45 and HLA-DR markers. For further details, all antigen-presenting cells are described in [Fig. 1].

[0048] Advantageously, antigen-presenting cells (APCs) are selected from dermal cDCl cells, dermal Langerin cDC2 cells, dermal Langerin+ cDC2 cells, and Langerhans cells (LCs). The detailed profile of markers expressed by these cells is given in Table 1 below.

[0049] [Tables 1] Dermal cDCl marker cells HLA-DR+, CD141++, CDlc / low, CDllclow, CD14, L angerin, CADMU, Clec9a+, CDlc+ cDC2 Langerin+ dermal HLA-DR+, CDla+, CDllc+, CD141, SIRPa+, Langér ine+, DC-SIGN, CDllb+, CDlc+ Langerhans cells (LCs) HLA-DR+, CDla++, Langerin+, CDllcweak, CDlc+, Birbeck granules+, E-Cadherin+, DC-SIGN, EpCAM+ Activation markers refer to all the markers described in the

[0050]

[0051]

[0052]

[0053]

[0054] Table 2. An antigen-presenting cell is considered activated when at least 2 activation markers are overexpressed in that cell, preferably at least 3 activation markers and, particularly preferably, at least 5 activation markers. Overexpression of an activation marker is defined as an increase in its expression of at least 20%, and preferably at least 30%, compared to the baseline expression level. Such an increase is measured between the antigen-presenting cells of two skin expiants from the same donor, only one of which was injected with the composition whose vaccine potential is to be determined, and the other with a control composition (water for injection or PBS). Step ic) of determining the possible migration of antigen-presenting cells within the skin expiry is also carried out by well-known immunohistochemical methods as described previously.

[0055] To do this, one determines whether or not the antigen-presenting cells are relocated near blood and / or lymphatic vessels. Determining such relocation can be done simply since blood and lymphatic vessels have characteristic structures that are easily identifiable within the histological section of the skin sample.

[0056] By relocalization of antigen-presenting cells in the vicinity or not of blood and / or lymphatic vessels, we mean an increase of at least 10% in the population of antigen-presenting cells in the vicinity of blood and lymphatic vessels, preferably of at least 20%.

[0057] Step ii) of determining the vaccine potential of the composition can then be simply carried out with regard to the result of steps ib) and, possibly ic).

[0058] Thus, it will be possible to determine whether the composition has vaccine potential with regard to an increase in the activation of antigen-presenting cells.

[0059] Thus, a composition associated with an activation of at most 10% of the antigen-presenting cells will have a low, or even zero, vaccine potential.

[0060] Conversely, a composition associated with an activation of at least 40% of the antigen-presenting cells will exhibit a high vaccine potential.

[0061] Preferably, an increase of at least 20% on at least two of the activation markers (HLA-DR, CD80, CD86, CD83, CD40 and CCR7) is observed in antigen-presenting cells in the case of a composition associated with high vaccine potential.

[0062] Finally, a composition associated with an intermediate activation of antigen-presenting cells and not exceeding either of the two previous thresholds will present an intermediate, or even moderate, vaccine potential.

[0063] Thus, it will also be possible to specify the vaccine potential of the composition with regard to a possible migration of antigen-presenting cells.

[0064] Thus, a composition associated with a relocation of at most 2% of the antigen-presenting cells near blood and / or lymphatic vessels will have a low, or even zero, vaccine potential.

[0065] Conversely, a composition associated with a relocation of at least 10% of the antigen-presenting cells to the vicinity of blood and / or lymphatic vessels will exhibit high vaccine potential.

[0066] For control purposes, the same steps ia), ib), ic) may be carried out with a negative control (e.g. PBS) and / or with a composition comprising a positive control corresponding to a composition known to have high vaccine potential.

[0067] According to a preferred embodiment, the method further comprises a step id) determination of the expression profile of genes related to the immune system within the skin expiry.

[0068] By way of examples of genes related to the immune system, and in a non-limiting manner, the following genes may be cited:

[0069] ABCB1, ABCF1, ABL1, ACKR4, ADA, ADGRE5, AHR, AICDA, AIRE, ALAS1, APP, ARG1, ARG2, ARHGDIB, ATG10, ATG12, ATG16L1, ATG5, ATG7, ATM, B2M, B3GAT1, BATF, BATF3, BAX, BCAP31, BCL10, BCL2, BCL2L11, BCL3, BCL6, BID, BLNK, BST1, BST2, BTK, BTLA, C1QA, C1QB, C1QBP, CIR, C1S, C2, C3, C4A / B, C4BPA, C5, C6, C7, C8A, C8B, C8G, C9, CAMP, CARD9, CASP1, CASP10, CASP2, CASP3, CASP8, CCL11, CCL13, CCL15, CCL16, CCL18, CCL19, CCL2, CCL20, CCL22, CCL23, CCL24, CCL26, CCL3, CCL4, CCL5, CCL7, CCL8, CCND3, CCR1, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL2, CD14, CD160, CD163, CD164, CD19, CD1A, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD27, CD274, CD276, CD28, CD34, CD36, CD3D, CD3E, CD3EAP, CD4, CD40, CD40LG, CD44, CD45R0, CD45RA, CD45RB, CD46, CD48, CD5, CD53, CD55, CD58, CD59, CD6, CD7, CD70, CD74, CD79A, CD79B, CD80, CD81, CD82, CD83, CD86, CD8A, CD8B, CD9, CD96, CD99, CDH5, CDKN1A, CEACAM1, CEACAM6, CEACAM8, CEBPB, CFB, CFD, CFH, CFI, CFP, CHUK, CIITA, CISH, CLEC4A, CLEC4E, CLEC5A, CLEC6A,CLEC7A, CLU, CMKLR1, CRI, CR2, CRADD, CSF1, CSF1R, CSF2, CSF2RB, CSF3R, CTLA4, CTLA4-TM, CTNNB1, CTSC, CTSG, CTSS, CUL9, CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL2, CXCL8, CXCL9, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CYBB, DEFB1, DEFB103A, DEFB103B, DEFB4A, DPP4, DUSP4, EBI3, EDNRB, EEF1G, EGR1, EGR2, ELP1, ENTPD1, ​​EÛMES, ETS1, FADD, FAS, FCAR, FCER1A, FCER1G, FCGR1A / B, FCGR2A, FCGR2A / C, FCGR2B, FCGR3A / B, FCGRT, FKBP5, FN1, FOXP3, FYN, G6PD, GAPDH, GATA3, GBP1, GBP5, GFI1, GNLY, GP1BB, GPI, GPR183, GUSB, GZMA, GZMB, GZMK, HAMP, HAVCR2, HFE, HLA-A, HLA-B, HLA-C, HLA-DMA, HLA-DMB, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HPRT1, HRAS, ICAM1, ICAM2, ICAM3, ICAM4, ICAM5, ICOS, ICOSLG, IDO1, IFI16, IFI35, IFIH1, IFIT2, IFITM1, IFNA1 / 13, IFNA2, IFNAR1, IFNAR2, IFNB1, IFNG, IFNGR1, IFNL1, IFNL2, IFNL2 / 3, IGF2R, IKBKB, IKBKE, IKBKG, IKZF1, IKZF2, IKZF3, IL10, IL10RA, IL11RA, IL12A, IL12B, IL12RB1, IL13, IL13RA1, IL15, IL16, IL17A, IL17B,IL17F, IL18, IL18R1, IL18RAP, IL19, ILIA, IL1B, IL1R1, IL1R2, IL1RAP, IL1RL1, IL1RL2, IL1RN, IL2, IL20, IL21, IL21R, IL22, IL22RA2, IL23A, IL23R, IL26, IL27, IL2RA, IL2RB, IL2RG, IL3, IL32, IL4, IL4R, IL5, IL6, IL6R, IL6ST, IL7, IL7R, IL9, ILF3, IRAK1, IRAK2, IRAK3, IRAK4, IRF1, IRF3, IRF4, IRF5, IRF7, IRF8, IRGM, ITGA2B, ITGA4, ITGA5, ITGA6, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITLN1, ITLN2, JAK1, JAK2, JAK3, KCNJ2, KIR3DL1, , KIR3DL2, KIR3DL3, KIR_Activating_Subgroup_l, KIR_Activating_Subgroup_2, KIR_Inhibiting_Subgroup_l, KIR_Inhibiting_Subgroup_2, KIT, KLRA1P, KLRB1, KLRC1, KLRC2, KLRC3, KLRC4, KLRD1, KLRF1, KLRF2, KLRG1, KLRG2, KLRK1, LAG3, LAIR1, LAMP3, LCK, LCP2, LEF1, LGALS3, LIF, LIERAI, LILRA2, LILRA3, LILRA4, LILRA5, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LIT AF, LTA, LTB4R, LTB4R2, LTBR, LTF, LY96, MAF, MALT1, MAP4K1, MAP4K2, MAP4K4, MAPK1, MAPK11, MAPK14, MAPKAPK2, MARCO, MASP1, MASP2, MBL2, MBP, MCL1, MIF, MME, MRI, MRC1, MS4A1, MSR1, MUC1, MX1, MYD88, NCAM1, NCF4, NCR1, NFATC1, NFATC2, NFATC3, NFIL3, NFKB1, NFKB2, NFKBIA, NFKBIZ, NLRP3, NOD1, NOD2, NOS2, NOTCH1, NOTCH2, NT5E, OAZ1, PAX5, PDCD1, PDCD1LG2, PDCD2, PDGFB, PDGFRB, PECAM1, PIGR, PLA2G2A, PLA2G2E, PLAAT4, PLAU, PLAUR, PML, POLR1B, POLR2A, POU2F2, PPARG, PPBP, PPIA, PRDM1, PRF1, PRKCD, PSMB10, PSMB5, PSMB7, PSMB8, PSMB9, PSMC2, PSMD7, PTAFR, PTGER4, PTGS2, PTK2, PTPN2, PTPN22, PTPN6, PTPRC, PYCARD, RAF1, RAG1, RAG2, RELA, RELB,RORC, RPL19, RTRAF, RUNX1, S100A8, S100A9, S1PR1, SDHA, SELE, SELL, SELPLG, SERPING1, SH2D1A, SIGIRR, SKI, SLAMF1, SLAMF6, SLAMF7, SLC2A1, SMAD3, SMAD5, S0CS1, S0CS3, SPP1, SRC, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, STING1, SYK, TAGAP, TAL1, TAPI, TAP2, TAPBP, TBK1, TBP, TBX21, TCF4, TCF7, TFRC, TGFB1, TGFBI, TGFBR1, TGFBR2, THY1, TICAM1, TIGIT, TIRAP, TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TNF, TNFAIP3, TNFAIP6, TNFRSF10C, TNFRSF11A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF17, TNFRSF1B, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13B, TNFSF15, TNFSF4, TNFSF8, TOLLIP, TP53, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TUBB, TYK2, UBE2L3, VCAM1, VTN, XBP1, XCL1, XCR1, ZAP70, ZBTB16, ZEB1 and sCTLA4. ,

[0070] Step ü) of determining the vaccine potential of the composition will then, in connection with this step id), specify the vaccine potential of the composition.

[0071] Preferably, the genes related to the immune system are cytokines.

[0072] Step ii) of determining the vaccine potential of the composition will then, in connection with this step id), determine the inflammatory potential associated with this same composition.

[0073] Examples of usable cytokines include interleukins and their receptors. Examples of interleukins include IL-1A, IL-1B, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17A, IL-17C, IL-17F, IL-19, IL-21, IL-22, IL-23, IL-27, IL-31 and IL-33, and examples of interleukin receptors include IL-10RA, IL-10RB, IL-1R1, IL-5RA (CD125) and IL-9R.

[0074] Chemokines, which are chemotactic cytokines that control the migration patterns and positioning of immune cells, as well as their receptors, can also be mentioned as usable cytokines. Examples of cytokines include C5, Eotaxin, MCP-4, TARC, MCP-1, MIP-3A, CCL22, CCL23, MIP-1B, RANTES, MCP-3, MCP-2, CX3CL1, IL8RA, INP10, L8RB, and CXCL3. Chemokine receptors include CCL13 (MCP-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CX3CR1, CXCR1, and CXCR2.

[0075] It should be noted that other cytokines (besides interleukins and chemokines) are conceivable as markers of inflammation. Examples of such cytokines include MCP-1, GM-CSF, TNFSF5, MCSF, GCSF, TNFSF6, IFNA2, IFNG, TNFA, TNFB, MIF, NAMPT, TRAIL, and IFNA1.

[0076] Advantageously, cytokines are selected from GM-CSF, MIP-la, MCP-1, IL-2, IL-4, IL-6, IL-8, IL-13, IL-12, IL-15, IL-16, MIP-3a, IP-10, MlP-la, MlP-lb, MDC, IL-27, and Eotaxin-3.

[0077] The cytokine expression profile can be determined within the skin expiry, the culture medium or even within the matrix.

[0078] Now, the inventors have highlighted that the cytokine expression profile within the matrix showed both greater diversity and higher levels of expression.

[0079] Advantageously, therefore, the determination of the cytokine expression profile is carried out within the skin expiry matrix.

[0080] According to a preferred embodiment, the method further includes a step id) determination of the level of mast cell degranulation within the skin expiry.

[0081] Step ii) of determining the vaccine potential of the composition will then, in connection with this step ie), determine again the inflammatory potential associated with this same composition.

[0082] Step ie) of determining the level of mast cell degranulation can be carried out using techniques well known to those skilled in the art. Techniques that can be used for this step include ELIS A or colorimetric techniques that allow the measurement of the presence of inflammatory mediators contained in mast cell granules, such as histamine or tryptase, or secreted de novo such as lipid mediators or cytokines / chemokines, or even fluorescence, immunofluorescence or fluorochrome techniques specific to mast cell granules.

[0083] Advantageously, this step ie) is carried out within a maximum of 6 hours following step ie) of administration, preferably within a maximum of 4 hours.

[0084] According to a preferred embodiment, step ie) of determining the level of mast cell degranulation within the skin expiry is carried out by fluorescence analysis.

[0085] Advantageously, this step ie) of determining the level of mast cell degranulation uses avidin.

[0086] Avidin is indeed a glycoprotein that binds very specifically to heparin contained in mast cell granules (THARP et al., J. Histochem. Cytochem vol. 33, pp. 27–32, 1985). Thus, during the degranulation process, the granules, once externalized, become directly accessible to avidin. Furthermore, in the case of tissue fixation, intracellular granules are accessible to avidin once tissue permeabilization occurs. This avidin can be complexed with a fluorochrome (avidin-FITC, avidin-Alexa488, avidin-sulfurhodamine 101, or any other fluorescent molecule) or with a bioluminescent molecule. It is also possible to use avidin alone (uncomplexed) and in combination with a complementary molecule.Examples of such a molecule include biotin complexed with a fluorochrome, a bioluminescent molecule, or any other identifiable molecule.

[0087] Now, determining the level of mast cell degranulation can use other markers, particularly those of the nucleus or plasma membrane, to facilitate the identification of granulocytes released from mast cells. It will also be possible to measure tryptase, histamine, beta-hexosaminidase, chymase, or any other molecules preformed within mast cell granules and released during degranulation in the culture medium.

[0088] Typically, the determination of the level of mast cell degranulation within the skin expiry can be carried out by following the protocol described in GAUDENZIO et al. (J. Clin. Invest., vol.126, p:3981-3998, 2016)

[0089] Advantageously, step ie) of determining the level of mast cell degranulation will be carried out on at least one histological section taken from the skin expiry.

[0090] To achieve this, well-known immunohistochemical methods can be used, which involve fixing the skin sample, embedding the sample (e.g., paraffin, OCT, EPON) before preservation, and finally preparing histological sections of the embedding block. Details of such methods are described, for example, in "Immunohistochemistry: Basics and Methods" by Igor Buchwalow (Springer Publishers).

[0091] The usable histological sections may have a very significant thickness, up to 500 µm. Advantageously, the histological section will thus have a thickness between 1 and 500 µm. Now, it is possible to use sections featuring more classic dimensions with a thickness between 2 and 25 µm.

[0092] As regards the determination of the level of mast cell granulation within the expiry itself, for each identified mast cell, it is determined whether it is associated with weak, moderate, or strong degranulation, and then the proportion (percentage) of mast cells associated with each of these types of degranulation (weak, moderate, and strong). The level of degranulation can also be analyzed automatically using image analysis software, a computer algorithm, or artificial intelligence techniques known as "machine learning" or "deep learning".

[0093] In detail, a mast cell with a low level of degranulation corresponds to a mast cell with 0 to 2 granules around it (or to a cell with a smooth outline); a mast cell with a moderate level of degranulation corresponds to a mast cell with 3 to 6 granules around it (or to a cell with an outline with a granular appearance); and a mast cell with a high level of degranulation corresponds to a mast cell with more than 6 granules around it (or to a cell with an exposed shape).

[0094] Thus, it will be possible to determine whether the composition has an inflammatory potential with regard to the percentage of mast cell degranulation as determined at the end of step ie).

[0095] Thus, a composition associated with a proportion of mast cells exhibiting more than 50% a low level of degranulation and / or less than 10% a high level of degranulation will present a low, or even zero, inflammatory potential.

[0096] Conversely, a composition associated with a proportion of mast cells exhibiting more than 50% of a high level of degranulation will present a high inflammatory potential.

[0097] The following examples are given solely to illustrate the subject matter of the present invention and do not in any way constitute a limitation. Examples

[0098] 1-Preparation of skin expiants

[0099] Skin expiants are prepared from complete skin samples taken from various donors, which samples included the epidermis, dermis, and hypodermis (1.5 to 2 cm). The expiants (epidermis, dermis, and hypodermis) were then cut using a metal punch to obtain cylinders 11 to 20 mm in diameter, in which the hypodermis thickness was adjusted to the desired value (0.5 to 1 cm). Finally, these expiants were kept afloat in a buffered saline solution until the "inclusion" step in the solidified matrix. This The inclusion step was performed using a procedure similar to that used for the NATIVES KIN™ model. Briefly, the skin expiry is gently deposited onto an insert (MILLICELL™ 8-well cup) with a porous membrane (PET, 1 µm porosity) at the bottom, containing a blood plasma-derived solution treated with an anticoagulant agent with reversible properties in the presence of calcium ions (sodium citrate). This solution contains 42% blood plasma, 50% of a 0.9% NaCl solution, 8% of a 1% CaCl2 saline solution, an antifibrinolytic agent (tranexamic acid or aprotinin), and 0.7% low-melting-point molten agarose (Agarose LMP GIBCOBRL, LIFE TECHNOLOGIES) (oven-melted at 65.5 °C). The antifibrinolytic agent functions to inhibit enzymes that can degrade the plasma matrix, these enzymes being secreted by the skin expiratory tissue, and thus to maintain the integrity of the expiratory tissue.

[0100] 2-Skin antigen-presenting cells and expiratory

[0101] 2.1-Identification of antigen-presenting cells within the skin expiry

[0102] The presence of antigen-presenting cells within the expiry was analyzed during culture and in the days following culture using several techniques: immunohistochemistry coupled with artificial intelligence (multiplexed imaging), single-cell transcriptomic analysis, and flow cytometry. Single-cell transcriptomic analysis revealed the presence, in the model and compared to human skin, of comparable proportions of immune cells (expressing PTPRC), stromal cells (expressing VIM), melanocytes (expressing MIANA), keratinocytes (expressing KRT14 and KRT1), and adipocytes over a 10-day period. Any changes in the transcriptome of all these cells were investigated over time.Of the 2,000 genes detected during the experiment, only a slight variation of just 55 genes in the immune compartment was detected between day 0 and day 5 of culture, with a return to normal between day 5 and day 10 of culture. These data indicate that the structural and immune cells contained in the HYPOSKIN models remain phenotypically and functionally stable for a period of at least 10 days.

[0103] In conclusion, the results unexpectedly showed that antigen-presenting cells within the skin expiry exhibit a number and diversity similar to those observed within skin in vivo. Moreover, the results also showed that this number and diversity of antigen-presenting cells is maintained from the first to the tenth day of culture.

[0104] 2.2-Demonstration of maturation of antigen-presenting cells within the skin expiry

[0105] In view of the previous result, the inventors questioned the maturation capacity of the antigen-presenting cells within the skin expiry.

[0106] Also, and to test this maturation capacity, several techniques were used: immunohistochemistry coupled with artificial intelligence (multiplexed imaging), single-cell transcriptomic analysis and flow cytometry.

[0107] Here again and unexpectedly, the results showed that in addition to preserving their number and diversity, the antigen-presenting cells also exhibit an intact maturation capacity.

[0108] Therefore, the skin expiry can be used to test the ability of an antigenic composition to activate antigen-presenting cells. At the same time, and as previously demonstrated by the inventors, it is possible to test the inflammatory potential associated with this same composition.

[0109] 3-Confirmation of the vaccine potential of the INFLUVAC TETRA composition

[0110] 125 pL of an INFLUVAC TERTR vaccine solution were injected into the tissue Adipose tissue was extracted from skin explants from 3 separate donors using a 27G syringe and a 12 mm long needle. As a negative control, 100 µL of water was injected into the adipose tissue of skin explants from the same donors.

[0111] The expiants were then cultured (incubator at 37°C, 5% CO2 and water-saturated atmosphere) for 8h, 24h and 48h.

[0112] Hematoxylin and eosin staining was performed on skin excipients from each donor before injection and 48 hours after injection of water or the vaccine composition. At the same time, the presence of any DNA fragmentation was tested on these same skin excipients.

[0113] The results showed, for the 3 donors, no alteration of cellular integrity 48h after injection of the vaccine composition.

[0114] After evaluating the stability and viability of the model during the study, cytokine production by the models was investigated. To obtain optimal results, the assay was performed on the culture medium, matrix, and lysate of the models injected with water and vaccine at 8 hours of culture for the three donors in the study.

[0115] Many cytokine concentration values ​​are not reported for the lysate because their concentration is below the detection limit of the MSD instrument. Regarding the assay on the culture medium, it allows for the measurement of the concentration of most cytokines in the panel, but in significantly lower quantities than the assay on the matrix, even though the concentration trends are the same for both assays. Consequently, the matrix will be used for future cytokine assays, as it allows for the detection of most cytokines at sufficient concentrations.

[0116] Following optimization of the MSD assay, the concentrations of 36 cytokines were measured in the matrix of study models. The uninjected models appeared to be the least inflamed and generally exhibited the lowest cytokine concentrations. At 8 hours post-injection, a slight increase in cytokine concentration was observed. A marked increase in cytokine production was observed in the 3 donors tested 24 hours post-injection, with higher cytokine concentrations in the vaccinated models, notably IFN-γ, TNF-α, IL-α, IP-10, eotaxin-3, IL-12p40, IL-13, IL-15, IL-16, MCP-4, MlP-α, MlP-β, MIP-3α, IL-β, GM-CSF, MCP-1, and TARC.

[0117] Finally, cytokine concentration increases slightly 48 hours after injection, particularly in vaccinated models. Overall, a biological response is observed following vaccination through the production of cytokines and chemokines linked to immune system activation in the models over time, notably with a peak 24 hours after injection.

[0118] In order to determine the presence of antigen-presenting cells, skin expiants from each of the donors before injection, 8h, 24h and 48h after injection of water or vaccine composition were cryopreserved at -80°C for immunohistochemical analysis, or embedded in paraffin for mast cell degranulation analysis.

[0119] Cryopreserved sections were then labeled with different antibodies specific to antigen-presenting cells (anti-CD45, CD207, CDlc, CD40, CCR7, CD80, CD86, CD83, and HLA-DR).

[0120] Analysis of these sections showed that injection of the vaccine composition leads to the activation of antigen-presenting cells (APCs) within the expiry, as well as the migration of a number of them. Consequently, the results therefore show that the administered vaccine is associated with the establishment of adaptive immunity.

[0121] Paraffin-embedded sections were used simultaneously to determine the level of mast cell degranulation within the tissue under different conditions. This was done by labeling with avidin coupled to a fluorochrome, which allows for the detection of mast cell granules. The first step involves deparaffinizing and rehydrating the fixed, paraffin-embedded sections. The sections were incubated for 30 minutes at room temperature in citrate buffer pH 6, then saturated and permeabilized for 40 minutes at 37°C with a 0.1% goat serum and Triton solution. The sections were then incubated for one hour at room temperature in a humid chamber with 5 µg / mL of avidin-sulforhodamin 101 (Avidin TEXAS RED; MERCK). Cell nuclei were then stained by incubating the sections with DAPI (D9542, SIGMA) at a dilution of 1 / 1000 for 3 minutes at room temperature. Mounting medium is added and a coverslip is placed on the sections. The slides are then analyzed under a fluorescence microscope to determine the level of mast cell degranulation in the different excipients.

[0122] The results of these degranulation experiments are presented in Table 3.

[0123] [Tables3] Percentage of granulocytes with water. Vaccine composition. Low degranulation level: 15%. Intermediate degranulation level: 30%. High degranulation level: 60%. Inflammatory potential: 30%.

[0124] The results made it possible to determine the level of degranulation of the mast cells identified in the samples and, after integration, to deduce the inflammatory potential of the injected composition. In this case, the results show that the administered vaccine is not associated with an inflammatory risk due to mast cell degranulation.

[0125] 4-Confirmation of the vaccine potential of the INFLUVAC TETRA composition

[0126] Analysis of the INFLUVAC TETRA vaccine composition revealed that this vaccine induces the expression of a cytokine signature indicative of cutaneous immune system activation, including the expression of GM-CSF, MlP-1a, MCP-1, IL-2, IL-4, IL-6, IL-8, IL-13, IL-12, IL-15, IL-16, MIP-3a, IP-10, MlP-1a, MlP-1b, MDC, IL-27, and Eotaxin-3 between 8 and 24 hours of culture. Using multiplex imaging, it was demonstrated that the vaccine induces the activation of dermal dendritic cells and Langerhans cells via an increase in the expression of the maturation / activation markers CD40, CCR7, CD80, CD86, CD83, and HLA-DR.

Claims

Demands

1. An in vitro method for determining the vaccine potential of a composition comprising the steps of: ia) transcutaneous administration to penetrate the skin barrier, which can be done subcutaneously, transdermally or topically, of the composition to a skin expiant, comprising the epidermis, dermis and epidermal appendages as well as a thickness of at least 5 millimeters of hypodermis, which expiant has been included, with the exception of the epidermis, in a liquid matrix capable of solidifying, then positioned within an insert the bottom of which is made of a porous membrane, allowing the liquid matrix to be prevented from passing through it before its solidification, and the diameter of which is between 5 and 40 mm; ib) determination of the activation status of antigen-presenting cells within the skin expiant by monitoring the expression of activation markers within these cells;and ii) determination of the vaccine potential of the composition with regard to an increase in the activation of antigen-presenting cells;

2. The method according to claim 1, characterized in that the antigen-presenting cells are selected from dermal cDCl cells, dermal cDC2 Langerin cells, dermal cDC2 Langerin+ cells and Langerhans cells.

3. The method according to claim 1 or 2, characterized in that step ia) consists of subcutaneous or transdermal administration of the composition.

4. The method according to any one of claims 1 to 3, characterized in that an antigen-presenting cell is considered activated when at least 2 activation markers are overexpressed in that cell, which activation markers are chosen from those described in Table 2. [Table 2]

5.

6.

7.

8.

9. Activation markers of antigen-presenting cells CD40 CCR7 CD86 CD83 CD80 HLA-DR The process according to claim 4, characterized in that the overexpression of an activation marker corresponds to an increase in its expression of at least 20%. The method according to any one of claims 1 to 5, characterized in that step ii) of determining the vaccine potential of the composition is carried out with regard to the proportion of antigen-presenting cells activated at the end of step ia), with: * a composition associated with an activation of at most 10% of the antigen-presenting cells will have a low, or even zero, vaccine potential; * A composition associated with activation of at least 40% of antigen-presenting cells will exhibit high vaccine potential. The method according to claim 1 or 2, characterized in that it further comprises a step ic) of determining the possible migration of antigen-presenting cells within the skin expiry. The method according to any one of claims 1 to 3, characterized in that it further comprises a step id) of determining the expression profile of cytokines within the skin expiry and in that step ii) further allows determining the inflammatory potential associated with this same composition. The method according to the preceding claim, characterized in that the determination of the cytokine expression profile is carried out within the skin expiry matrix.

10. The method according to any one of claims 1 to 3, characterized in that it further comprises a step ie) of determining the level of mast cell degranulation within the skin expiry and in that step ii) further allows the determination of the inflammatory potential associated with this same composition.