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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/461—Cellular immunotherapy characterised by the cell type used
  • A61K39/4615—Dendritic cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
  • A61K39/4622—Antigen presenting cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/46—Cellular immunotherapy
  • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
  • A61K39/464838—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/998—Proteins not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• New adjuvant immunity compound compositions containing it and methods using said adjuvant compound
• the present invention relates to the field of immunity adjuvant compounds, that is to say compounds capable of inducing an increase in the immune response against an antigen, in order to increase the effectiveness of the stimulation of the immune response by an immunogenic composition, or also to increase the preventive or therapeutic effectiveness of a vaccine composition.
• the immunity adjuvant compounds or compositions are useful for improving the conditions for stimulating an immune response against antigens.
• the immunity adjuvant compounds or compositions are used to increase the quantity of antibodies produced against a given antigen, or to increase the quantity of effector T cells produced, whether they are T cells helper (T helper) or T-cytotoxic cells.
• T helper T cells helper
• T-cytotoxic cells T cells helper cells helper cells
• the association of an antigen with an immunity adjuvant compound or composition in addition to increasing the level of the immune response, by greater production of antibodies or effector T cells specific for the antigen also makes it possible to reduce the quantity of antigen included in an immunogenic or vaccine composition, and, if necessary, to reduce the frequency of injection of said immunogenic or vaccine composition.
• an immunity adjuvant with the antigen of interest is in particular required when the immunogenicity properties of this antigen of interest, when administered without an adjuvant, are insufficient to stimulate an immune response. effective given the immunization objectives pursued.
• the immunity adjuvant compounds or compositions induce a better immune response against a antigen of interest in separate ways.
• Certain adjuvants of immunity act on the immune system by inducing a more efficient production of antibodies against the antigen of interest, for example by activating macrophages, dendritic cells, B cells and T cells, or by improving the conditions under which the antigen of interest is presented to the various immuno-competent cells.
• Compounds or adjuvants of immunity can increase the immune response by prolonging the duration of release of the antigen of interest, by increasing the quantity of antigen absorbed by the cells presenting the antigen, by positively regulating the antigen processing by these cells, by stimulating the release of cytokines, by stimulating isotypic switching and the maturation of B cells and / or by eliminating immunosuppressive cells.
• T Helper helper T lymphocytes
• T-cytotoxic lymphocytes (“CTL” or “CD8 + ” cells) are of great interest for the development of immunogenic compositions or effective vaccine compositions.
• the cells of the immune system which prepare antigens by fragmenting them into peptides, then by presenting these peptides, in association with the molecules of the major histocompatibility complex (MHC) of class I or class II, are essentially the macrophages and the cells. dendritic.
• the dendritic cells are capable of priming the antigens, then of presenting the peptides resulting from the coating of the antigens to the naive T cells. Dendritic cells activate T cells more effectively than any other cell presenting the antigen.
• Dendritic cells are generally present in the body at locations exposed to foreign antigens, such as the skin, liver, intestine, blood and lymphoid tissue. Overall, dendritic cells are classified according to whether they are at an immature stage or at a mature stage. Mature dendritic cells are able to efficiently capture and process antigens by endocytosis, and also to express high levels of co-stimulatory molecules, such as the CD40, CD80 and CD86 molecules, as well as the Complex molecules. Major in Histocompatibility (CMH) HLA-DPv.
• CMH Histocompatibility
• dendritic cells express the CD83 marker and secrete high levels of various cytokines and chemokines which act as aids in the activation of T cells. Besides their role in activating naive T cells, dendritic cells mature can also influence the balance of the Th1 / Th2 immune response. Various studies have indicated that dendritic cells preferentially activate Th1-type responses, presumably due to the secretion of IL-12 by activated dendritic cells. (acatonia et al., 1995, J. Immunol., vol. 154: 5071; Hilkens et al., 1997, blood, vol. 90: 1920).
• dendritic cells can induce the generation of clones of Th1 or Th2 cells.
• the important role played by dendritic cells in the presentation of the antigen and the activation of T cells has aroused great interest with a view to using or activating dendritic cells in immunotherapy.
• the activation or obtaining of mature dendritic cells is of particular importance in the field of vaccines and immunotherapy for cancers.
• nucleic acids containing CpG type oligonucleotide sequences are capable of inducing the maturation of dendritic cells.
• a new family of adjuvant immunity compounds has been characterized, of the peptide type, which activate dendritic cells and induce the maturation of immature dendritic cells.
• the adjuvanting immunity compounds according to the invention are derived from the head domain (knob) of the "fiber" protein of the capsid of an adenovirus.
• the subject of the invention is an adjuvant immunity compound consisting of: - a polypeptide (i) comprising an amino acid sequence of 30 amino acids in length contained in the “head” domain of the “fiber” protein ”Of the capsid of an adenovirus, said amino acid sequence comprising the chain of amino acids forming the double-layered structure ⁇ designated“ EF ”contained in said“ head ”domain; or - a peptide (ii) analog of said polypeptide (i) whose amino acid sequence comprises, with respect to the sequence of said polypeptide (i), at least one substitution or at least one deletion of an amino acid, said peptide analog retaining said structure in double ⁇ sheet designated "EF".
• a polypeptide (i) comprising an amino acid sequence of 30 amino acids in length contained in the “head” domain of the “fiber” protein ”Of the capsid of an adenovirus, said amino acid sequence comprising the chain of amino acids forming the double-layered structure ⁇ designated“ EF ”con
• the invention also relates to an adjuvanting composition of immunity comprising an adjuvanting compound as defined above, in association with at least one physiologically compatible excipient. It also relates to an immunogenic composition as well as to a vaccine composition comprising an adjuvant compound as defined above, in combination with at least one antigen of interest.
• the invention also relates to a method for the in vitro maturation of immature human or animal dendritic cells, characterized in that it comprises the following steps: a) cultivating in vitro a population of cells enriched in immature human or animal dendritic cells , in an appropriate culture medium; and b) incubating the cells cultivated in step a) with an adjuvant compound or an adjuvant composition as defined above, for a time sufficient to induce the maturation of the dendritic cells.
• the invention also relates to a population of cells enriched in mature dendritic cells, capable of being obtained by the above maturation process.
• the subject of the invention is also an adjuvant composition of immunity, characterized in that it comprises a population of mature dendritic cells obtained according to the above process of maturation.
• the invention also relates to a method for stimulating T cells specific for the antigen in vitro, characterized in that it comprises the following steps: a) obtaining a population of cells enriched in mature dendritic cells by the maturation process above; b) bringing the population of cells enriched in mature dendritic cells obtained in step a) into contact with a population of cells enriched in T cells originating from the same individual, man or animal. DESCRIPTION OF THE FIGURES
• Figure 1 diagram of the structure of the fiber protein in virions of adenovirus serotype 5, respectively wild ( Figure 1A) and viruses devoid of the "head” domain ( Figure 1B).
• the fiber protein of the adenovirus comprises three structural domains, starting from the N-terminal end to the C-terminal end: The “tail” which is linked non-covalently to the "penton base”part; the “stem” and the “head” domain binding to the CAR receptor, which is responsible for the attachment of adenovirus to permissive cells.
• the wild-type adenovirus Ad5 serotype has “fibers” comprising a long “stem” having 22 repeats of the ⁇ -sheet motif.
• the “stem” of the fiber protein is shorter, with seven repeats of the ⁇ -leaf motif and ends with a trimerization pattern followed by an "opal” stop codon.
• Figure 2 Effect of adenovirus capsid components on the phenotype of dendritic cells.
• the dendritic cells were cultured in the culture medium alone (NS; unstimulated dendritic cells), or in the presence of the purified proteins of the adenovirus Ad5 (1 ⁇ g per 10 6 cells), or of LPS (1 ⁇ g / ml) ; then were characterized for their expression of the marker CD11c and markers of specific maturation of the dendritic cells.
• Figure 2A Dendritic cells cultured with Hx and Pb (fiber-bound penton base) isolated from HeLa cells infected with the Ad5 adenovirus.
• Hx and Pb fiber-bound penton base
• the dendritic cells were incubated in the absence of "unstimulated dendritic cells, NS" or in the presence of 1 ⁇ g of protein Fi or protein of the head domain (FIG. 3A), where the dendritic cells were incubated with determined quantities of proteins Fi and Hx (respectively 1 ⁇ g) and various concentrations of head proteins (in a range from 0.5 to 0.02 ⁇ g) ( Figures 3B, 3C). Dendritic cells and culture supernatants were collected and tested as indicated below. For FIG. 1A, the dendritic cells were characterized for the expression of the CD11c marker and the maturation markers. The percentages of CD11c + cells and the average immunofluorescence (MFI) values for the maturation markers were represented.
• MFI average immunofluorescence
• FIG. 4 proof of a direct interaction between the head domain protein and dendritic cells, and proof of the need for the head domain in the maturation of dendritic cells induced by the Ad5 adenovirus.
• dendritic cells purified using magnetic beads coated with anti-CD11c antibodies were stimulated with 0.5 ⁇ g of protein from the head domain; dendritic cells purified unstimulated (NS) were used as controls.
• NS dendritic cells purified unstimulated
• MFI average immunofluorescence
• the histograms show the average immunofluorescence (ivIFI) values for the markers of CD11c + cell maturation.
• the results presented are representative of two independent tests.
• Figure 5 absence of expression of the CAR receptor on dendritic cells and low permissiveness of dendritic cells vis-à-vis infection with adenovirus serotype 5.
• Immature dendritic cells isolated before stimulation were incubated with ascites fluid containing monoclonaur antibodies. anti-CAR (bold line), or with unrelated ascites fluid (gray line).
• the dendritic cells were analyzed by flow cytometry (FACS).
• the HeLa and CHO cells were used as positive and negative controls respectively.
• Figure ⁇ mapping of the region of the head domain of the fiber involved in the maturation of dendritic cells.
• Figure 6A schematic representation of the conformational structure of the head domain of the adenovirus Ad5 fiber protein (Xia et al., 1994). This diagram shows the different regions A to J having a ⁇ sheet structure as well as the respective link loops AB, CD, DG, GH, Hl and IJ. The upper line indicates the numbering of the amino acids, starting from the N-terminal methionine residue of the complete fiber protein of the adenovirus Ad5. The head domain regions of the fiber protein interacting with the CAR receptor are shown as solid black boxes on the bottom line of the figure.
• FIG. 6B is a linear representation of the different deletion mutants of the head domain of the fiber protein of the adenovirus Ad5.
• the header domain sequence is indicated by solid boxes; deleted regions are indicated as thin lines.
• stimulating cells with purified allogenic TCD4 + cells were used, respectively, unstimulated dendritic cells (NS), dendritic cells stimulated with headless domain extracts (MS; “mock - stimulated "), or dendritic cells stimulated with the wild head domain protein (" k ⁇ ob T ”) or with deletion mutants of the head domain of the fiber protein.
• NS unstimulated dendritic cells
• MS headless domain extracts
• dendritic cells stimulated with the wild head domain protein (“ k ⁇ ob T ”
• the results presented are averages of three separate trials.
• FIG. 7 the dendritic cells stimulated with the head domain of the fiber protein of the adenovirus ⁇ d ⁇ stimulates in vivo TCD8 + cells specific for the antigenic peptide GP33 derived from the glycoprotein of LCiWiV.
• Figure TA illustrates the rejection of donor cells by recipient mice vaccinated with dendritic cells.
• B6 mouse splenocytes labeled with CFSE (5-6-carboxyflu ⁇ rescei ⁇ diacetate succinimidyl ester) were loaded with the peptide GP33, then the treated splenocytes were transfused into recipient B6 mice which were previously immunized with, respectively, (i ) dendritic cells stimulated with the head domain of the fiber protein (respectively 2.5 ⁇ 10 5 , 9 ⁇ 10 4 and 3 ⁇ 10 4 cells), loaded with GP33 or (ii) with dendritic cells loaded with GP33 (2.5 ⁇ 10 5 cells) stimulated with Pb or Hx, or non-stimulated (NS), or (iii) with dendritic cells loaded with NP366 (2.5 x 10 5 ) stimulated with the leader domain of the fiber protein, or (iv) with the GP33 peptide in emulsion in incomplete Freund's adjuvant (IFA).
• IFA incomplete Freund's adjuvant
• FIG. 7B represents the secretion of IFN- ⁇ ex vivo by the same mice as for FIG. 7A.
• Freshly isolated spleen cells and peripheral blood lymphocytes (PBL) were incubated for 20 hours with the peptide GP33; evaluation of the number of specific cells of GP33 secreting IFN- ⁇ by an Elispot IFN-y test :; the results are expressed in SFC ("spot forming colony") / 10 5 CD8 + T cells (test average carried out in triplicate + SD). Each spot corresponds to a cell secreting IFN- ⁇ .
• FIG. 1 represents the secretion of IFN- ⁇ ex vivo by the same mice as for FIG. 7A.
• Freshly isolated spleen cells and peripheral blood lymphocytes (PBL) were incubated for 20 hours with the peptide GP33; evaluation of the number of specific cells of GP33 secreting IFN- ⁇ by an Elispot IFN-y test :; the results are expressed in SFC ("spot forming colon
• FIG. 7C illustrates the cytolytic activity of effector cells derived from mice vaccinated with dendritic cells (3 ⁇ 10 4 cells) and not stimulated or stimulated with the leader domain of the fiber protein of the adenovirus Ad5.
• the spleen cells were incubated with the target cells EL4 labeled with 51 Cr and then loaded with the peptide GP33 or the peptide NP366, in a chromium release test only the activated ones specific to GP33 will lyse targets ( 51 Cr).
• the head domain of the capsid fiber protein of an adenovirus is capable of interacting directly with immature dendritic cells and of causing their activation and their maturation in mature dendritic cells.
• Adenoviruses appear as icosahedral particles 70 to 100 nanometers in diameter, depending on the serotype.
• the viral capsid comprises two major constituent elements, respectively, (i) the hexon (Hx) which forms the faces and the penton (Pn) located at the 12 vertices.
• the base of the penton (or penton-base) is associated with the fiber protein, which is a spicular projection emanating from the base of the penton, consisting of a trimer of polypeptide IV.
• Fiber is a protein of 581 to 587 amino acid residues for long-fiber adenovirus species, such as species C and A, and 319 to 325 amino acid residues for the shortest fiber, such as adenoviruses of serotypes 3 and 7, belonging to species B.
• the fibers are all formed from three distinct structural domains, respectively the tail, the stem and the head.
• the tail fits into the penton-base.
• the stem consists of the periodic repetition of a pattern of fifteen residues, each forming a ⁇ -sheet structure. The number of repeats of the unitary pattern defines the length of the stem, which is variable between the fibers of different species of adenovirus.
• the head which is formed of a trimer of a polypeptide sequence of 180 to 200 amino acid residues.
• each head monomer associates a skeleton of eight anti-parallel ⁇ sheets linked together by loops whose conformation varies considerably according to the serotypes.
• a polypeptide consisting of a fragment of the head domain of the fiber protein of a adenovirus, said fragment comprising the amino acid sequence forming the anti-parallel ⁇ double-sheet structure designated “EF” induces the activation and maturation of immature dendritic cells.
• a peptide fragment of the head domain of the fiber protein of an adenovirus comprising the amino acid sequence delimiting the antiparallel ⁇ -sheet and deleted in other peptide regions of the head domain, like for example the anti-parallel ⁇ sheet "Hl"
• a polypeptide consisting of the head domain of the fiber protein and which comprises the deletion of the amino acid sequence forming the antiparallel ⁇ -sheet structure has lost the activation and maturation properties immature dendritic cells of the complete head domain.
• a polypeptide consisting of the head domain and comprising the deletion of two amino acids in the F region of the ⁇ antiparallel EF sheet does not have the activation and maturation properties of immature dendritic cells which are observed with a polypeptide comprising the complete amino acid sequence of the head domain.
• the results obtained by the applicant show that a polypeptide consisting of a peptide fragment of the head domain of the fiber protein of an adenovirus, and which comprises the amino acid sequence forming the double-layered structure ⁇ antiparallel EF, said polypeptide not comprising the amino acid sequences forming other ⁇ -sheet structures contained in the head domain, is capable of inducing the activation and the maturation of immature dendritic cells.
• the results obtained by the applicant show that the activation and maturation properties of immature dendritic cells of the head domain of the fiber protein of an adenovirus are carried by a small peptide region of said head domain, the amino acid regions forming the double-layered structure ⁇ EF.
• the leader domain of the fiber protein of all adenoviruses has a common structure consisting of a succession of ⁇ sheets linked together by peptide loops, similar to those of the leader domain of adenovirus serotype 5 (Ad5) shown in the Figure 6A.
• Ad5 adenovirus serotype 5
• the applicant has also shown that, at least from the point of view of the activation and maturation properties of immature dendritic cells, this structural identity of the head domain of all the adenoviruses also implies a functional identity.
• an adenoviral vector comprising chimeric fiber proteins consisting of a tail and a stem originating from an adenovirus of serotype 5 and a head domain originating from an adenovirus of serotype 3 is also capable of inducing the activation and maturation of immature dendritic cells.
• the above results have enabled the applicants to define a new family of adjuvanting immunity compounds, derived from the head domain of the fiber protein of an adenovirus, this new family of adjuvanting immunity compounds constituting a first object of the invention.
• the subject of the invention is an adjuvanting immunity compound consisting of: - a polypeptide (i) comprising an amino acid sequence of 30 amino acids in length contained in the “head” domain of the protein t fiber 5> de the capsid of an adenovirus, said amino acid sequence comprising the chain of amino acids forming the double-layered structure ⁇ designated "EF s> contained in said" head "domain; or - a peptide (ii) analog of said polypeptide (i) whose amino acid sequence comprises, with respect to the sequence of said polypeptide (i), at least one substitution or at least one deletion of an amino acid, said analog peptide retaining said structure in double ⁇ sheet designated "EF".
• an adjuvant compound corresponding to the above definition induces the maturation of immature dendritic cells.
• an adjuvant compound above induces the expression by the dendritic cells of the class I and class II molecules of the MHC, as well as markers specific for the mature dendritic cells, such as the markers CD40, CD80 and CD86.
• Dendritic cells stimulated by an adjuvant compound as defined above induce the proliferation of allogeneic CD4 + T lymphocytes in mixed lymphocyte reaction (MLR) tests and also induces the secretion of IL-12 and TNF ⁇ in a dose dependent manner .
• An adjuvant compound according to the invention acts directly on immature dendritic cells without attachment to the CAR receptor.
• said polypeptide (i) has an amino acid sequence of at least 30 amino acids in length since the polypeptide (i) comprising in all cases a amino acid sequence of 30 amino acids in length containing the amino acid sequence forming the ⁇ EF sheet of the head domain of the fiber protein of an adenovirus, which carries the function of maturation of immature dendritic cells.
• the amino acid sequence of the ⁇ EF sheet can comprise, with respect to the amino acid sequence of the ⁇ EF sheet of the head domain of the adenovirus fiber protein from which it is derived, a or several substitutions of an amino acid.
• the amino acid substitution (s) in the sequence of the ⁇ EF sheet are such that they do not modify said ⁇ -sheet structure.
• the amino acid sequence forming the ⁇ EF sheet of an analogous peptide (ii) is identical to the amino acid sequence of the ⁇ EF sheet of the parent polypeptide (i).
• the chain of amino acids forming the double-sheet structure ⁇ EF is located approximately at the center of the amino acid sequence of said polypeptide.
• the amino acid sequence of the ⁇ EF sheet of the head domain of the fiber protein of the adenovirus of serotype 5 has a length of 8 amino acids.
• this amino acid sequence begins at the amino acid residue at position 479 and ends at the amino acid residue located at position 486 of the complete fiber protein of serotype 5 adenovirus
• an adjuvant compound consisting of a polypeptide (i) having the minimum length of 30 amino acids
• the amino acid sequence of the ⁇ EF sheet, of 8 amino acids is preceded, at the N-terminal end, by a sequence of 11 amino acids in length corresponding to a part of the DE loop and is followed, on the C-terminal side, by a sequence of 11 amino acids in length comprising a part of the FG loop.
• the amino acid sequence of the ⁇ EF sheet contained in an adjuvant polypeptide (i) according to the invention is located "approximately" in the center of the amino acid sequence of the polypeptide (i) when the sequences located respectively on the N- side terminal and C-terminal side of the sequence of the ⁇ EF sheet do not have an identical length.
• the length of the sequences located respectively on the N-terminal side and on the C-terminal side of the amino acid sequence of the ⁇ EF sheet can differ by a length of up to 20 amino acids, one relative to to the other.
• said polypeptide (i) comprises, from the N-terminal end to the C-terminal end, the amino acid sequences of the DE loop , the ⁇ EF sheet and the FG loop.
• said adjuvant compound consists of the polypeptide whose amino acid sequence begins at amino acid residue at position 463 and ends at amino acid residue at position 515 of the complete fiber protein sequence.
• an adjuvant compound according to the invention of the polypeptide type (i) has a length of at least 30 amino acids, and of at most 195 amino acids and very preferably of at most 180 amino acids. .
• an adjuvant compound according to the invention of the polypeptide type (i) has a length of at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190 or 195 amino acids in length.
• an adjuvanting immunity compound consisting of a polypeptide (i) as defined above
• the sequence of said polypeptide (i) with a length of "n” amino acids, consists into a sequence of “n” consecutive amino acids of a corresponding sequence contained in the head domain of the fiber protein of the adenovirus considered.
• the polypeptide (i), with a length of "n” amino acids comprises a sequence of "" consecutive amino acids of the head domain and comprising the sequence of the ⁇ EF sheet as well as one or more two additional amino acid sequences, the total length of the additional amino acid sequences being "n - x" amino acids, located at the N-terminus and / or at the C-terminus of the sequence head domain of "x" amino acids. It should be understood that “n” is an integer between 30 and 195 and that "x" is an integer between 30 and "nx”.
• the additional sequence or sequences may consist of sequences of peptides detectable by specific antibodies of such a peptide, which could therefore be used as a marker.
• the additional sequences can consist of sequences allowing easier purification of the adjuvant compound after its synthesis, whether by chemical synthesis or by synthesis by genetic recombination.
• the additional sequences are chosen from polyhistidine sequences, for example sequences comprising from 4 to 10, and preferably 6, histidine residues.
• an adjuvant compound according to the invention of the polypeptide type comprises an amino acid sequence of the head domain of the fiber protein of an adenovirus which comprises, from the N-terminal end towards the C-terminus, the ⁇ -D sheet.
• an adjuvant component according to the invention, of the polypeptide type (i), comprises an amino acid sequence of the head domain which comprises, from the end
• an adjuvant compound of the polypeptide type (i) comprises, the N-terminal end towards the C-terminal end, respectively the ⁇ D sheet, the DE peptide loop, the ⁇ EF sheet, the FG peptide loop and the ⁇ G sheet.
• such a polypeptide (i) comprises the amino acid sequence starting at the amino acid at position 454 and ending at the amino acid at position 521 of complete fiber protein.
• the polypeptide (i) or the analogous peptide (ii) also comprises, in addition to the amino acid sequence of the fiber head domain, also the last repeat subunit of the fiber rod.
• Such a peptide adjuvant compound according to the invention comprising the last repeating subunit of the fiber rod is capable of possessing great structural stability and of blocking the part of the head domain in its native conformation and in the form of a trimer. peptide.
• a peptide adjuvant compound of the invention which comprises a polypeptide (i) derived from the fiber protein of adenovirus of serotype 5
• said polypeptide (i) comprises the acid sequence amino acids starting at the amino acid residue at position 380 and ending at the amino acid residue at position 581 of the amino acid sequence of the complete fiber protein.
• the polypeptide (i) comprises part of the head domain of the fiber protein of an adenovirus which is a human adenovirus.
• the human adenovirus is chosen from adenoviruses of subgroup B, which comprises adenoviruses Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34 and Ad35, or from adenoviruses of subgroup C, which comprises adenovirus Ad1, Ad2, Ad5 and Ad6.
• the human adenovirus is chosen from the group consisting of adenoviruses of serotypes 12 18, 31, 3, 7, 11, 14, 16, 21, 34, 35, 1, 2, 5, 6, 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 4, 40 and 41.
• an adjuvant peptide according to the invention derives from the head domain of the fiber protein.
• the complete fiber proteins of the adenoviruses Ad5, Ad3 and Ad12 are represented as the amino acid sequences SEQ ID No 1, SEQ ID No 2 and SEQ id No 3, respectively.
• the leader domain of the adenovirus Ad5 fiber protein begins at the amino acid residue at position 400 of the sequence SEQ ID No. 1.
• the head domain of the adenovirus Ad3 fiber protein begins at the amino acid residue at position 132 of the sequence SEQ ID No. 2.
• the leader domain of the adenovirus Ad12 fiber protein begins at the amino acid residue at position 409 of the sequence SEQ ID No. 3.
• the polypeptide (i) comprises an amino acid sequence chosen from the following sequences: - the sequence starting with acid amino at position 463 and ending with amino acid at position 515 of the sequence SEQ ID No. 1 - the sequence starting with amino acid at position 195 and ending with amino acid at position 247 of the sequence SEQ ID N ° 2. - the sequence starting with amino acid in position 472 and ending with amino acid in position 535 of the sequence SEQ ID N ° 3.
• a particular embodiment of the adjuvant compound according to the invention consists of a peptide (ii) analogous to the polypeptide (i) defined above, the amino acid sequence of which comprises, with respect to the sequence of said polypeptide (i), at least one substitution or at least one deletion of an amino acid é, said analog peptide retaining the double-sheet structure ⁇ EF.
• the substitution (s) or deletion (s) of an amino acid with respect to the sequence of the polypeptide
• the adjuvant analog peptide (ii) comprises 2,3,4,5,6,7,8,9 or 10 substitutions or deletions of an amino acid, relative to the amino acid sequence of the polypeptide (i ) parent.
• said adjuvant compound is characterized in that the polypeptide (i) or the analogous peptide (ii) consists of a cyclic polypeptide.
• the adjuvant peptides (ii) which include in their sequence one or more amino acid differences compared to the corresponding sequence contained in the head domain of the natural fiber protein of the adenovirus considered, nevertheless has adjuvant properties, that is ie properties of inducing the maturation of immature dendritic cells, of the same order of magnitude as the parent polypeptide (i) from which it derives.
• the invention relates to adjuvant peptides derived from the head domain of the fiber protein of an adenovirus which has the same adjuvant activity as the adjuvant peptides specifically described in the present description.
• an adjuvant compound according to the invention of inducing the maturation of immature dendritic cells can be easily verified by those skilled in the art, for example by determining the level of expression MHC class I or class II molecules, or the level of expression of a specific marker for the maturation of dendritic cells, such as the molecules CD40, CD80 and CD86.
• an adjuvant compound according to the invention to stimulate the production of IL-12 and TNF ⁇ by dendritic cells.
• an amino acid contained in the sequence of the parent polypeptide (i) is substituted by an amino acid of the same amino acid class, among the classes of acid amino acids (D, E), basic (K, R, H), non-polar (A, V, L, I, P, M, F, W) or even uncharged polar amino acids (G, S, T, Y , N, Q).
• the “percentage identity” between two amino acid sequences is determined by comparing the two optimally aligned sequences, through a comparison window.
• the part of the amino acid sequence in the comparison window can thus include additions or deletions (for example “gaps”) with respect to the reference sequence (which does not include these additions or deletions) so to obtain an optimal alignment between the two sequences.
• the percentage of identity is calculated by determining the number of positions at which an identical amino acid residue is observed for the two sequences compared, then by dividing the number of positions at which there is identity between the two amino acid residues by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of amino acid identity of the two sequences between them.
• the optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
• a peptide adjuvant compound according to the invention can be synthesized by conventional methods of synthetic chemistry, either homogeneous chemical syntheses in solution or in solid phase.
• a person skilled in the art can use the polypeptide synthesis techniques in solution described by HOUBEN WEIL (1974).
• a peptide adjuvant compound according to the invention can also be chemically synthesized in the liquid or solid phase by successive couplings of the different amino acid residues (from the N-terminal end to the C-terminal end in liquid phase, or the C-terminal end towards the N-terminal end in solid phase).
• Those skilled in the art can in particular use the solid phase peptide synthesis technique described by Merrifield (1965a; 1965b).
• a peptide adjuvant compound according to the invention can be synthesized by genetic recombination, for example according to a production process comprising the following steps: (a) preparing an expression vector into which a nucleic acid encoding the peptide adjuvant compound of the invention has been inserted, said vector also comprising the regulatory sequences necessary for the expression of said nucleic acid in a chosen host cell; (b) transfecting a host cell with the recombinant vector obtained in step (a); (c) culturing the host cell transfected in step b) in an appropriate culture medium; (d) recovering the culture supernatant from the transfected cells or the cell lysate of said cells, for example by sonication or by osmotic shock; and (e) separating or purifying, from said culture medium, or from the cell lysate pellet, the recombinant peptide adjuvant compound of the invention.
• a recombinant peptide adjuvant compound of the invention can in particular refer to the techniques for preparation of the recombinant vectors, for cell transfection and for purification which are described in the examples. Most preferably, a baculovirus type vector is used to infect Sf9 cells, as described in the examples.
• a person skilled in the art can advantageously implement techniques of purification described by iVlolinier-Frenkel (2002), by
• any of the adjuvant peptide compounds according to the invention can be used in combination with an antigen against which an immune response is sought.
• a peptide adjuvant compound according to the invention can be combined, with a view to inducing an immune response, in humans or animals, either with a peptide antigen or with a carbohydrate type antigen, for example a carbohydrate type antigen identical or similar, from the point of view of its antigenic recognition, to an antigen specifically expressed by tumor cells.
• the peptide adjuvant compound according to the invention can be combined with any type of antigen against which an immune response is sought.
• a peptide adjuvant compound of the invention is chemically coupled to one or more antigens against which an immune response is sought, in the form of an adjuvant peptide / antigen peptide conjugate or also in the form of 'an adjuvant peptide-carbohydrate antigen conjugate.
• the present invention also relates to an immunogenic conjugate consisting of an adjuvant peptide compound according to the invention, which is covalently linked to an antigen against which an immune response is sought.
• the subject of the invention is also an adjuvant composition of immunity comprising a peptide adjuvant compound as defined above in the present description, in association with at least one physiologically compatible excipient.
• the adjuvant compound and the antigen are linked directly to each other covalently, for example via a peptide bond - CO-NH-.
• a peptide conjugate is preferred in which the adjuvant compound and the antigen are separated from each other, within said conjugate, by a spacer chain.
• the adjuvant compound and the antigen are separated from each other, within said conjugate, by a spacer chain chosen from SMCC or SIAB, which are all both of the bifunctional compounds.
• SIAB described by Hermanson GT (1996, Bioconjugate techniques, San Diego: Académie Press, pp 239-242), is the compound of formula (I) below:
• the SIAB compound comprises two reactive groups, respectively an iodoacetate group and a Sulfo-NHS ester group, these groups reacting respectively on amino and sulfhydryl groups.
• the SMCC compound which is described by Samoszuk M.K. et al. (1989, Antibody, Immunoconjugates Radiopharm., 2 (1): 37-46), is the compound of formula (II) below:
• the immunogenic conjugate comprises a spacer chain consisting of a linear spacer peptide.
• a linear spacer peptide preferably having from 3 to 30 amino acids in length, advantageously from 5 to 20 amino acids in length and most preferably from 7 to 15 amino acids in length will be chosen.
• the linear spacer peptide is essentially, if not exclusively, made up of positively or negatively charged amino acids at pH 7.0 in order to increase the overall hydrophilicity of said immunogenic peptide conjugate.
• the spacer peptide is characterized in that it consists of a poly (lysine) chain consisting of 3 to 30 lysine residues, advantageously from 5 to 20 and very preferably from 7 to 15 length lysine residues.
• the adjuvant compound and the antigen are separated from each other, within said peptide conjugate, by a spacer chain consisting of a spacer peptide branched, preferably an oligodendrimeric poly (lysine) structure, as described for example by Basak et al. (1995).
• said peptide conjugate can comprise several copies respectively of the adjuvant compound and / or of the antigen per molecule of conjugate, advantageously from 2 to 8 copies of the adjuvant compound and / or the antigen, preferably at most 4 of the adjuvant compound and / or the antigen, per conjugate molecule.
• the present invention also relates to a peptide adjuvant compound as defined above in the description, for its use as an adjuvant active ingredient of an immunogenic composition or of a vaccine composition.
• immunogenic composition a composition containing a peptide adjuvant compound as defined above in association with at least one antigen against which a cell-mediated immune response is sought, in order to produce specific antibodies against said antigen
• vaccine composition a composition containing an adjuvant peptide compound as defined above, in combination with at least one antigen against which a cell-mediated immune response is sought in order to prevent or in order to treat a disease, in particular a disease caused by a pathogenic agent of viral, fungal or bacterial type or even a tumor.
• the invention also relates to the use of an adjuvant compound peptide as defined above for the manufacture of an immunogenic or vaccine composition.
• an immunogenic composition or a vaccine composition comprising a peptide adjuvant compound as defined above, in combination with at least one antigen.
• the antigen can be used in the form of a mixture with the adjuvant peptide compound of the invention.
• the adjuvant peptide compound and the antigen included in a vaccine composition or in an immunogenic composition of the invention are in the form of an immunogenic conjugate as defined above.
• dendritic cells stimulated with a peptide adjuvant compound according to the invention and loaded with a determined antigen in this case the peptide GP33 of LGMV S was capable of inducing, in the animal, a cytotoxic immune response by stimulation of CD8 + T cells specific for the GP33 antigen. It has been shown in particular that mature dendritic cells stimulated by a peptide adjuvant compound according to the invention and presenting the antigen of interest to the cells of the immune system were capable of inducing an immune response of the cytotoxic type. causing the rejection of splenocytes having on their surface the antigen of interest.
• an immunogenic composition or in a vaccine composition according to the invention any type of antigen can be used since, in all cases, and whatever the antigen or antigens, the adequate peptide of the invention will exercise its activity activation of dendritic cells.
• an immunogenic composition or a vaccine composition according to the invention comprises an amount ranging from 10 nanograms to 1 milligram of a peptide adjuvant compound as defined above, preferably from 100 nanograms to 100 micrograms of said adjuvant peptide compound, and most preferably from 100 nanograms to 10 micrograms of said adjuvant peptide compound.
• antigens capable of being included in an immunogenic composition or in a vaccine composition according to the invention in combination with a peptide adjuvant compound, mention may be made of bacterial antigens derived in particular from B. pertussis,
• the antigens can also be viral antigens such as antigens derived from poliovirus viruses, adenovirus, para influenza virus, respiratory syncytial virus, influenza virus, encephal ⁇ myélitis virus, Newcastle disease virus, pox virus, virus
• the antigens may also consist of antigens originating from any allergen, such as allergens derived from extracts of flower or grass pollen, allergens purified from household dust, etc.
• allergens derived from extracts of flower or grass pollen, allergens purified from household dust, etc.
• viral antigens of interest there are also cited the antigens derived from papillomavirus proteins, in particular human papillomaviruses, and in particular the proteins L1, E6 and E7, in particular strains HPV-16.
• Other illustrative viral antigens consist of antigens derived from proteins of the human immunodeficiency virus (HIV), in particular HIV-1, and most preferably derived from the ENV protein of the HIV-1 virus.
• HIV human immunodeficiency virus
• antigens of interest capable of being included in an immunogenic composition or in a vaccine composition according to the invention consist of tumor antigens, that is to say antigens expressed by cancer cells, whether these antigens are peptide or carbohydrate in nature.
• the immunogenic compositions or the vaccine compositions according to the invention find their use in particular in the treatment, both curative and preventive, of cancers, in particular of cancers induced by viruses such as for example ATL (acute T leukemia) caused by the HTLV1 virus, or cervical cancer caused by the papillomaviruses, or burkitt lymphoma or Kaposi's sarcoma caused by viruses of the Herpes family, respectively Epstein-barr (EBV) and HHV8, as well as in the treatment of AIDS or to prevent or treat allergic inflammatory reactions.
• ATL acute T leukemia
• cervical cancer caused by the papillomaviruses
• burkitt lymphoma or Kaposi's sarcoma caused by viruses of the
• the subject of the invention is also a method for immunizing a man or an animal, more specifically a mammal, against an antigen of interest, said method comprising a step during which one administers to humans or animals an immunogenic composition or a vaccine composition as defined above.
• an immunogenic composition or a vaccine composition according to the invention which is in a form suitable for systemic or mucosal administration, for example intranasally, sufficient to be therapeutically effective for a subject in need of such treatment.
• An immunogenic composition or a vaccine composition according to the invention is in solid or liquid form, in particular in the form of an oil-in-water emulsion in which the antigen or antigens of interest is (are) scattered.
• an immunogenic composition or a vaccine composition according to the invention in the form of an oil-in-water emulsion, a person skilled in the art can use an SPT type emulsion as described on page 147 of the book “Vaccine Design, The subunit and adjuvant approach ”, [M. POWELL, M. Newman Ed., Plénum Press, (1995)] as well as the emulsion MF59 described on page 183 of the same work.
• physiologically compatible excipient within the meaning of the invention is meant a liquid or solid bulking agent, a diluent or any other substance which is not physiologically active and which is of great safety for the patient and which can be used for systemic administration. or local, for example on the mucous membranes, of an immunogenic composition or a vaccine composition according to the invention.
• physiologically acceptable excipients are described in detail including the 4 lem edition "2002" of the Pharmacopoeia
• the invention also relates to a process for the in vitro maturation of human or animal immature dendritic cells in which immature dendritic cells are stimulated with a peptide adjuvant compound as defined in the present description.
• a further subject of the invention is therefore a process for the in vitro maturation of human or animal immature dendritic cells, characterized in that it comprises the following steps: (a) cultivating in vitro a population of cells enriched in human immature dendritic cells or animal, in an appropriate culture medium; (b) incubating the cells cultivated in step (a) with a peptide adjuvant component or even with an adjuvant composition as defined in the present description, for a time sufficient to induce the maturation of the dendritic cells.
• the population of cells enriched in immature human or animal dendritic cells used in step a) of the process can be obtained from a sample of bone marrow or a sample of human or animal blood, according to well-defined techniques.
• step b) of the above maturation process the immature dendritic cells are incubated with a final concentration of the peptide adjuvant compound ranging from 10 nanograms per ml to 1 ⁇ g / ml, preferably ranging from 50 nanograms per ml to 1 mcg / ml.
• step b) of the maturation process the immature dendritic cells are incubated for a period ranging from 1 h to 48 hours, with the final concentration selected for the adjuvant peptide compound.
• the invention also relates to a population of cells enriched in mature dendritic cells loaded with an adjuvant compound or with an adjuvant composition as defined above.
• An adjuvant compound according to the invention can therefore be detected, for example using an antibody directed specifically against this adjuvant compound, either in the cytoplasm or on the membrane surface of these dendritic cells which are capable of being obtained by the maturation process as defined above.
• the mature dendritic cells obtained according to the above maturation process are characterized in that they simultaneously express the molecules of classes I and class II of the MHC as well as the specific markers of the dendritic mature cells CD40 CD80 and CD86.
• the present invention also relates to an adjuvant cellular composition of immunity, characterized in that it consists of a population of mature dendritic cells charged (i) with a adjuvant compound or with an adjuvant composition as defined above, and (ii) loaded with the antigen of interest capable of being obtained by the above maturation process.
• a dose of adjuvant cellular composition of immunity as defined above, for administration to patients comprises a number of mature dendritic cells ranging from 10 6 to
• the mature dendritic cells are preferably suspended in a saline liquid medium necessary for their survival for a few hours, preferably at least three hours. Most preferably, the mature dendritic cells are suspended in an appropriate culture medium comprising all of the nutrients allowing their long-term survival, for example for several days, preferably for at least 2 days.
• the invention also relates to a method for manufacturing an immunogenic cell composition, characterized in that it comprises the following steps: a) cultivating in vitro a population of cells enriched in immature human or animal dendritic cells, in a medium of appropriate culture; b) incubating the cells cultivated in step a) with an adjuvant peptide compound or else with an adjuvant composition as defined above, for a time sufficient to induce the maturation of the dendritic cells; c) adding to the cells cultivated in step b) at least one antigen of interest against which an immune response is sought.
• step c) of incubation of the dendritic cells with at least one antigen of interest can either be simultaneous with step b) in which the adjuvant peptide compound is incubated with the cells, or at otherwise be after step b).
• steps b) and c) are carried out simultaneously.
• a subject of the invention is also a method for manufacturing an immunogenic cell composition, characterized in that it comprises the following steps: a) cultivating in vitro a population of cells enriched in human or animal dendritic cells in an appropriate culture medium; b) incubate the cells cultivated in step a) with an immunogenic conjugate as defined above, for a time sufficient to induce the maturation of the dendritic cells.
• the final concentration added to the dendritic cells is variable depending on the nature and the molecular weight of the antigen of interest considered.
• LCMV peptide GP33 is added to dendritic cells.
• a person skilled in the art can adapt the final concentration which must be added in step c) (first method), or the final concentration of immunogenic conjugates which must be added in step b) the method (second method), thanks to its general technical knowledge concerning the loading of dendritic cells with an antigen of interest.
• the general conditions of the methods for manufacturing an immunogenic cell composition above are also identical to those used for the method of maturation of dendritic cells which has been described previously.
• the invention also relates to an immunogenic cell composition, characterized in that it comprises a population of mature dendritic cells loaded with the antigen of interest obtained by the methods for its manufacture which are described above.
• the mature dendritic cells loaded with the antigen of interest are characterized in that they express the class I and class II molecules of the MHC, as well as the specific markers of the mature dendritic cells CD40, CD80 and CD86.
• the invention also relates to a method for immunizing a human or animal body against an antigen of interest, which method comprises a step during which administers to the individual an adjuvant cellular composition as defined above, before, simultaneously, or after a step of administration of the antigen of interest.
• the subject of the invention is also a method for immunizing a human or animal body against an antigen of interest, which method comprises a step during which an immunogenic cell composition according to the invention is administered to the individual.
• the present invention is further illustrated by the following examples:
• A. MATERIAL AND ETHQDES A.1 Preparation of adenoviruses (Ad) and capsid proteins of adenovirus (Ad), including adjuvant peptide compounds according to the invention.
• the Ad5E1 adenovirus is a serotype 5 adenovirus defective for replication and deleted in both the early regions E1 and E3.
• the Ad5 E1 ° adenovirus carries wild type fiber proteins (WT) (iVlolinier-Frenkel et al., 2002).
• WT wild type fiber proteins
• the Ad5E1 ° ⁇ knob adenovirus, a deletion mutant for the fiber protein is derived from the Ad5E1 ° adenovirus by the insertion of a stop codon.
• FIG. 1 shows the schematic structure of the fibers of the adenoviruses Ad5E1 ° and Ad5E1 ° ⁇ knob.
• the Ad5E1 ° and Ad5E1 ° ⁇ knob virions were isolated by isopycnic ultracentrifugation in a continuous cesium chloride gradient (Molinier-Frankel et al., 2002).
• the Hexon capsid proteins (abbreviated as “Hx”) and the penton capsomers (abbreviated as “Pn”, for the penton base + fiber combination) were isolated from HeLa cells infected with the adenovirus.
• the penton-base proteins (also designated “Pb”), fiber (also designated “Fi”) and head domain of the wild type 5 adenovirus fiber were isolated in the form of recombinant proteins from infected Sf9 cells.
• Ad5 fiber proteins were also analyzed, carrying deletions of variable length in the head domain (Santis et al., 1999), respectively the mutant proteins designated FÎ ⁇ 402-480, Fi ⁇ HI, Fi ⁇ EF and FÎ ⁇ LT485 - 486 on Figure 6B. These mutated fiber proteins were produced as recombinant proteins in Sf9 cells.
• the adenovirus proteins were purified according to a protocol described by Molinier-Frenkel et al. (2002), Karayan et al. (1994) and Novelli et al. (1991).
• the adenovirus proteins were purified by a process comprising the following three steps: (i) precipitation with ammonium sulfate, (ii) high performance anion exchange liquid chromatography and (iii) concentration-ultrafiltration step using concentration membranes having a cutoff threshold of 100 kDa.
• the protein samples were analyzed by conventional techniques of SDS 1% -polyacrylarnide gel electrophoresis (Molinier-Frenkel et al., 2002) and immuno-imprinting (Karayan et al., 1994).
• A.2- Obtaining and culture of dendritic cells. The method was adapted from the technique described by Mayordomo et al. (1995). C57BU6 (H2 b ) mouse bone marrow cells (Harlan, Gannat, France) deleted in lymphocytes were cultured overnight in a complete culture medium (RPMI 1640 supplemented with 10% fetal calf serum (FCS) , 2 mM L- glutamine, 50 ⁇ iWl-mercaptoethanol, 100 U / ml of penicillin, 100 ⁇ g / ml of streptomycin).
• FCS fetal calf serum
• the non-adherent cells were removed and resuspended in complete culture medium in the presence of 2000 U / ml of recombinant GM-CSF (rGM-CSF, R & D System, Mineapolis, Minnesota) and 100 U / ml of Recombinant IL4 (rlL4, R & D System).
• the culture medium was replaced on day 4.
• aliquots of the non-adherent cells were resuspended at the density of 3 ⁇ 10 6 / ml in PBS buffer containing 1% FCS.
• the cells were then incubated for one hour at 37 ° C (i) without (unstimulated dendritic cells; NS-DG), or (ii) with capsid components of AdS, or (iii) with corresponding chromatographic fractions obtained from extracts of Sf9 cells infected with an empty baculovirus vector (dendritic cells stimulated by the empty vector; MS-DC), as indicated in the legends of the figures.
• the cell concentration was adjusted to 3 ⁇ 10 5 cells per ml with complete medium supplemented with GM-CSF. Control cells were incubated with 1 ⁇ g / ml of LPS from Eco // (sigma).
• the cells were recovered and analyzed by flow cytometry techniques, mixed lymphocyte reactions (MLRs) and immunization techniques. On Day 8, the culture supernatants were collected.
• the dendritic cells were purified using magnetic beads conjugated with a mouse anti-CD11c monoclonal antibody (Miltenyi Biotech). The dendritic cells were washed in PBS buffer containing 1% FCS.
• anti-FCII / IIIR antibody 24G2; Pharmingen
• the cells were incubated with various combinations of the following monoclonal antibodies (all marketed by Pharmingen): anti-lA b (AF6-120.1) and anti-CD40 (3/23), conjugated to PE anti-CD11c (HL3) and anti-CD80 (16-10A1) conjugated to FITC, anti-H2-D b (28-8-6) and anti-CD86 (GL1) biotinylated + streptavidin-PerCP (Becton Dickinson).
• Anti-fiber-tail mouse monoclonal antibodies have also been used (4D2.5; HONG et al., 1997).
• dendritic cells on Day 6 cells of the CHO line (ATCC No. CCL-61) and cells of the Hela line CCL-2 ATCc CCI2 and CHO were labeled with a monoclonal antibody originating anti-CAR ascites E1.1, as well as with control ascites.
• Counter staining was performed using biotinylated rat anti-mouse IgG and a streptavidin-PE conjugate.
• the flow cytometry analyzes were carried out on a F ⁇ CSCalibur device (Becton Dickinson).
• dendritic cells on Day 8 were distributed in increasing doses in culture wells and co-cultured for 4 days with aiiquot fractions of purified CD4 + splenocytes (2 x 10 5 cells / well) allogeneic (Balb / c, H2 d ).
• the proliferation of CD4 + T cells was measured by incorporation of 3 H-thymidine (1 ⁇ Ci / well) during the last 18 hours of the co-culture step.
• the cell culture supernatants Day 8 dendritics were tested using the ELISA kits IL12p70 and TNF ⁇ (Pharmingen).
• mice were immunized subcutaneously (sc) in the flank with 2.5 ⁇ 10 5 mature dendritic cells in the presence of the various capsid components of the adenovirus and then loaded with GP33 or NP366 (10 ⁇ M). Ten days later, the mice received an intravenous injection of 3 ⁇ 10 7 syngeneic splenocytes labeled with the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) as described by OEHEN et al. (1997).
• CFSE fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester
• the percentage of CFSE + donor cells in recipient splenocytes or peripheral blood lymphocytes (PBLs) was determined using flow cytometry analysis (FACS). Rejection of donor cells was calculated using the following formula: ratio of% CFSE + cells in immunized mice /% CFSE + cells in naive mice] x 100.
• A.8 T ⁇ sfe ELlSPOT-IFt Nitrocellulose microplates (Millipore) were coated with an anti-mouse IFN ⁇ rat antibody (R4-6A2; Pharmingen), then the wells were washed and saturated with complete medium. Aiquots of freshly isolated splenocytes (10 6 cells / well), or freshly isolated peripheral blood lymphocytes (PBLs) (2 x 10 5 cells / well), in triplicates, were added to the culture wells in complete medium containing 30 U / ml of recombinant human IL2 (hrlL2, Boehringer) and 10 "6 M of peptides GP33 or NP366.
• PBLs peripheral blood lymphocytes
• the cells secreting IFN ⁇ form spots there (SFC).
• SFC spot there
• the cells are counted according to the technique described by Molinier -Frenkel et al. (2002) The values obtained with the NP366 peptide were subtracted from the average of the values of the triplicate tests obtained with the GP33 peptide.
• EL-4 labeled with 51 Cr (10 -4 ⁇ Ci per cell) which were incubated with the peptide GP33 or NP366. After a culture of five hours at 37 ° C., the culture supernatants were collected and the radioactivity was measured using a device of the Top-Count type (Packard Instruments). In the control samples, the target EL4 cells were incubated with the medium alone in order to determine the level of spontaneous release of 51 Cr, and with 2% of alkyltrimethylammonium bromide (Sigma) in order to determine the total release of 51 Cr.
• EXAMPLE 1 Effect of the capsid components isolated from the adenovirus serotype 5 (Ad5) on the phenotype of the cendendium cells. Immature dendritic cells were incubated with penton (Pn) or hexon (Hx) capsomeres which were purified from HeLa cells infected with Ad5. The majority of dendritic cells stimulated with Hx exhibited a low level of mature phenotype, compared to unstimulated dendritic cells (NS-DC), with low expression of the class II molecules of CivIH, and the molecules CD40, CD80 (B7.1 ) and CD86 (B.7.2), as shown in Figure 2A.
• Pn penton
• Hx hexon
• the dendritic cells stimulated with the penton (Pn) massively expressed a mature phenotype, similar to the phenotype observed for the dendritic cells stimulated by LPS, with a high level of expression of the different markers, as illustrated in FIG. 2A.
• the adenovirus Pn capsomer consists of two structural entities, respectively penton base (Pb) and fiber (Fi)
• Pb penton base
• Fi fiber
• the complete recombinant proteins Pn and Hx were used as control samples.
• the results obtained indicate that the protein Fi is sufficient to reproduce the entire stimulating effect produced by the capsomers Pn, while the protein Pb alone does not induce any detectable effect on the maturation of the dendritic cells, as illustrated in the figure.
• E3CAMPLE 2 Role of the head domain of the fibr ⁇ in the maturation of the dendritic cells by the adenovirus and by the fiber protein.
• the adenovirus fiber comprises three structural domains, respectively, from its N-terminal end to its C-terminal end, the tail ("tail") the rod ("schaft") and the head ("Knob”).
• the head domain was expressed in the form of a recombinant protein, according to the techniques described by NOVELLI et al. (1991) and by Hong et al. (1997).
• the dendritic cells which were incubated with the head domain of the fiber expressed a mature phenotype, similar to that observed with the dendritic cells stimulated with the complete fiber protein Fi, as illustrated in FIG. 3A.
• Dendritic cells stimulated with the head domain of the fiber are capable of strongly inducing the proliferation of allogeneic CD4 + T cells in a mixed lymphocyte reaction (MLR) assay as illustrated in FIG. 3B.
• MLR mixed lymphocyte reaction
• the degree of maturation of the dendritic cells varies according to the increasing concentrations of protein of the head domain of the fiber (FIG. 3B).
• the dendritic cells stimulated by the head domain stimulating allogeneic CD4 4 T cells more effectively than the non-stimulated control dendritic cells (NS) or the dendritic cells stimulated with the Hx protein.
• NS non-stimulated control dendritic cells
• the mixed lymphocyte reaction (MLR) value for the 0.2 ⁇ g dose of head protein was similar to the value obtained with a 1 ⁇ g dose of fiber protein. This result is compatible with the fact that the amount of head protein present in a sample of 0.2 ⁇ g of head protein roughly corresponds to the amount of head protein contained in a sample of 1 ⁇ g of fiber protein.
• the head protein also induced the secretion of IL-12 and TNF ⁇ in a dose-dependent manner, as illustrated in FIG. 3C.
• the head domain was capable of directly targeting dendritic cells and inducing their maturation, without the participation of intermediate cells.
• CD11c + cells were purified using magnetic beads coated with anti-CD11c antibodies, the purified CD11c + cells having been used for maturation tests.
• the unstimulated dendritic cells are capable of increasing the expression of MHC molecules and of eostimulatory molecules, the dendritic cells stimulated with the head domain express a significantly more mature phenotype than the control cells (FIG. 3A).
• the recombinant protein Fi has been shown to be able to bind to immature dendritic cells, with 63% of cells positive on day 6 of culture (FIG. 3B).
• the cell-linked Fi protein was detected using an anti-tail monoclonal antibody, which implies that the epitope contained in the tail was accessible, and which suggests that attachment of the Fi protein to the cell surface was carried out via the head domain.
• the dendritic cells expressed a significantly less mature phenotype than after incubation with the Ad5E1 ° vector.
• the head domain of the fiber is the stimulating factor. common of all active viral components, and that the head domain carries the majority of the determinants responsible for the stimulation of murine dendritic cells observed with the Ad5 virions, the complete capsomer Pn, the complete fiber protein, and the recombinant protein of the head domain the insulated fiber.
• EXAMPLE 3 Absence of attachment of the head domain of the adenovirus fiber protein to the CAR receptor.
• dendritic cells do not express the CAR receptor.
• the expression of this crucial protein for binding the head domain to differentiated dendritic cells was analyzed by flow cytometry (FACS) under the culture conditions used in the present work.
• FACS flow cytometry
• HeLa cells which are known to express about 10,000-30,000 CAR cell molecules, and cells of the CHO line were used as the negative control as a positive control. No CAR expression could be detected above the background noise of control ascites on immature dendritic cells collected before stimulation, despite highly specific staining of HeLa cells, as illustrated in FIG. 5A.
• Tests have been performed to determine whether the mechanism of stimulation of dendritic cells by the head domain involves effective penetration of the adenovirus and expression of this gene.
• An adenoviral vector of serotype 5 encoding an “Enhanced Green Fluorescent Protein” (EGFP) protein with two infection index values (MOI) 10,000 and 30,000 was used.
• EGFP Enhanced Green Fluorescent Protein
• MOI infection index
• FIG. 4C an MOI index of 10,000 viral particles per cell in fact induces efficient maturation of the dendritic cells.
• significant expression of the fluorescent protein EGFP by dendritic cells was observed only at the dose of 30,000 particles per cell (FIG. 4C), which indicates a low degree of permissiveness to infection, by dentritic cell adenovirus.
• EXAMPLE 4 Mapping of the region of the domain® head of the fiber protein responsible for the maturation of dendritic cells.
• the induction of the maturation of the dendritic cells was tested with four mutants of adenovirus Ad5 fiber protein carrying deletions in the head domain (FIGS. 6A and 6B).
• the deletion present in the recombinant fiber protein ⁇ 402-481 covers the AB peptide loop, the ⁇ B and C sheets, the CD peptide loop as well as the N-terminal part of the DG peptide mouth.
• the mutated recombinant fiber protein Fi ⁇ HI is only devoid of the Hl peptide loop.
• Fi ⁇ EF and F ⁇ LT485-486 carry deletions in the short region of the double anti-parallel ⁇ EF sheet (Fi ⁇ EF), or a deletion of the two amino acid residues I485 and T486 which form the sheet ⁇ F (FÎ ⁇ LT485-486) (see KJrky et al., 2000 and Xia et al 1993).
• the mutated Fi proteins Fi ⁇ EF and Fi ⁇ LT 485-486 are in the form of trimeric fibers, while the mutated Fi proteins FÎ ⁇ 402-481 and ⁇ Hi are deficient in the trimerization of the fiber (see Santis et al., 1999).
• WT wild head domain
• MLR mixed lymphocyte reaction
• the three other mutated fiber proteins did not induce maturation of the dendritic cells, which suggests that the region in double antiparallel ⁇ -sheet EF, and more specifically the short ⁇ -sheet F , is essential for the maturing effect of dendritic cells.
• the dendritic cells stimulated with the head domain of the fiber protein were compared with dendritic cells stimulated with other components of the adenovirus, for their effectiveness in inducing CD8 + T cell response specific to the GP33 peptide derived from the LCMV glycoprotein, which is restricted to haplotype D b .
• the fluorescent vital dye CFSE was used to easily follow the presence of adoptively transferred splenocytes by flow cytometry analysis and by monitoring their elimination by specific CD8 + T cells induced by dendritic cell immunization. As illustrated in FIG. 7A, the population of spleen cells labeled with CFSE and loaded with the peptide GP33 decreased in 24 hours in the blood of all the mice immunized with the dendritic cells stimulated by the proteins Pb, Hx, or the protein of the head domain of the fiber and brought into contact with the peptide GP33.
• mice treated with dendritic cells stimulated with the head domain of the fiber significantly faster cell rejection was observed in mice treated with dendritic cells stimulated with the head domain of the fiber, with a rejection rate similar to that observed in mice immunized with the peptide GP33 emulsified in l Freund's incomplete adjuvant (IFA).
• the rejection curve of the cells in mice immunized with the dendritic cells stimulated with the head domain of the fiber is loaded with the peptide NP366 is identical to the curve observed in the mice immunized with the dendritic cells loaded with the peptide GP33 and put in presence of Pb or Hx, or even unstimulated. As illustrated in FIG.
• mature dendritic cells with the head domain of the fiber are ten times more effective in inducing rejection of syngeneic splenocytes than dendritic cells stimulated by the other components of adenovirus, as suggested by comparison of the effects obtained by immunizing with 3 ⁇ 10 4 dendritic cells stimulated by the head domain, to be compared with the effects obtained after immunization with 2.5 ⁇ 10 5 dendritic cells stimulated with Hx and Pb.
• analysis of CFSE + cells in the spleens of recipient mice, 10 days after the adoptive transfer provided even more significant results. 5% of CFSE + cells persisted in mice immunized with domain-stimulated dendritic cells.
• CFSE + cells represent 72% and 60% of initial CFSE + cells respectively in im mice equipped with dendritic cells stimulated respectively by the proteins Pb and Hx.
• Ten days after the adoptive transfer of the splenocytes labeled with CFSE (which corresponds to Day 20 after immunization with dendritic cells), the number of CD8 + T cells secreting IFNy was determined by carrying out ELISPOT tests ex vivo. As illustrated in FIG.

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