US20070059681A1 - Method for production of bioresorable microparticles, microparticles thus obtained and use thereof - Google Patents

Method for production of bioresorable microparticles, microparticles thus obtained and use thereof Download PDF

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US20070059681A1
US20070059681A1 US10/570,948 US57094804A US2007059681A1 US 20070059681 A1 US20070059681 A1 US 20070059681A1 US 57094804 A US57094804 A US 57094804A US 2007059681 A1 US2007059681 A1 US 2007059681A1
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microparticles
protein
ns3h
pla
bioresorbable
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Yasemin Ataman-Onal
Thierry Delair
Genevieve Inchauspe
Pascale Jeannin
Glaucia Paranhos-Baccala
Bernard Verrier
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Biomerieux SA
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Biomerieux SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to novel bioresorbable particles to which protein substances are bonded, that are useful in particular in the field of vaccination.
  • Protein substances such as proteins and peptides, also called antigens, are widely used in the treatment of many diseases, such as diseases of viral origin, generally in the form of a vaccine formulation.
  • compositions containing the antigens contain adjuvants.
  • vaccine formulations often comprise immunological adjuvants for enhancing cell-mediated responses and humoral responses.
  • oriented protection is intended to mean protection involving both the cell-mediated response and the humoral response of the immune system.
  • particulate carriers associated with antigens have been used, said antigens being either adsorbed onto the carrier, or trapped in the carrier.
  • Such carriers possess multiple copies of the antigen of interest presented to the immune system and promote the trapping and retention of the antigens in the local lymph nodes.
  • the particles can be phagocytozed by antigen-presenting cells and can increase antigen-presentation to the immune system.
  • examples of such carriers include poly(methyl methacrylate) polymers and also microparticles made of polylactides, such as poly(D- or L-lactic acid) known as D-PLA or L-PLA, respectively, and of poly(lactide-co-glycolide)s known as PLG.
  • PLG-based microparticles in which antigens are trapped are capable of giving an immune response.
  • Moore et al. (1995, Vaccine, 13:1741-1749) have shown that the microencapsulated HIV virus gp120 antigen induces an HIV-specific CD4+ and CD8+ T-cell response.
  • Vordemeier et al. (1995, Vaccine, 13-1576-1582) have shown that the PLG-trapped Mycobacterium tuberculosis antigen induces, in mice immunized with such an antigen, both a humoral response and a T-cell response.
  • microparticles which have the antigens adsorbed or grafted onto their surface (Rock K. L., Efficient MHC I presentation of exogenous Ag, PNAS 1993).
  • Some authors such as Eldrige et al., in Infect. Immun., 59:2978-2986 (1991), have, however, indicated that, in order to achieve an appropriate adjuvant effect, the antigens must be trapped in microparticles.
  • PCT patent application WO 97/02810 describes particles consisting of a biodegradable polymer, onto which antigens can be adsorbed. These particles are useful for the delivery of these antigens.
  • the drawback of these particles is their lamellar nature due to the use of crystalline or partially crystalline polymers, such that it is not possible to control their size.
  • particles intended for vaccination must have a submicronic size in order to be effective in transfection and in immunization.
  • Patent applications WO 98/33487 and WO 00/06123 describe, for their part, polylactide or PLG-based microparticles onto which antigens are adsorbed and the use thereof for stimulating immune responses. All the microparticles of these patent applications, onto which antigens are adsorbed, have been obtained using a surfactant in order to maintain the colloidal stability of said microparticles, and also a stabilizer, such as poly(vinyl alcohol), for the preparation of the polymer particles.
  • a surfactant in order to maintain the colloidal stability of said microparticles
  • a stabilizer such as poly(vinyl alcohol)
  • a first subject of the invention consists of a method for preparing bioresorbable microparticles to which protein substances are bonded, characterized in that it comprises the steps of:
  • microparticles obtained by means of the method of the invention are devoid of stabilizer and of surfactant such that they are novel.
  • Another subject of the invention consists of the bioresorbable microparticles to which protein substances are bonded, which are obtainable by the method of the invention.
  • the particles of the invention conserve their colloidal stability.
  • microparticles of the invention are useful for stimulating both a cell-mediated response and a humoral response, such that they are useful both in therapy and in diagnosis.
  • another subject of the invention consists of the use of the microparticles of the invention for preparing a medicinal product, and also the pharmaceutical compositions, in particular vaccines, comprising the microparticles of the invention.
  • Another subject of the invention consists of the use of the microparticles of the invention for the in vitro diagnosis of pathological states related to the protein substance bonded to said microparticles.
  • microparticles intended for vaccination must not be toxic for the organism that receives them, while at the same time conserving their colloidal stability.
  • the method of the invention that uses neither surfactant nor stabilizer, makes it possible, against all expectations, to obtain such particles.
  • microparticle is intended to mean particles of at most micronic, preferably at most submicronic, size so as to allow them to enter antigen-presenting cells.
  • At most micronic size is intended to mean a size of less than or equal to 999 ⁇ m, and the term “at most submicronic size” is intended to mean a size of less than or equal to 999 nm.
  • the microparticles have a particle diameter of less than or equal to 3 ⁇ m. More preferably, the particles of the invention are of submicronic size, with preferably a diameter of between 150 and 900 nm, more preferably between 250 and 700 nm.
  • the size of the particles is readily determined by techniques known to those skilled in the art, such as, for example, using scanning electron microscopy, quasi-elastic light scattering or transmission electron microscopy.
  • toxic microparticle is intended to mean a microparticle comprising at least one compound capable of causing biological disorders, such as metabolic disturbances, in the organism having received the microparticle.
  • the first step of the method of the invention consists of the preparation of said microparticles from at least one bioresorbable polymer without stabilizer and without surfactant.
  • bioresorbable polymer is intended to mean a polymer capable of degrading, in the organism into which it has been introduced, into compounds that can be eliminated via the natural pathways. This polymer may be amorphous, slightly crystalline or crystalline.
  • bioresorbable polymers include, without limitation, poly( ⁇ -hydroxylated acids), poly(hydroxybutyric acids), polycaprolactones, poly-orthoesters and polyanhydrides.
  • the bioresorbable polymer used in the method of the invention is a poly( ⁇ -hydroxylated acid) such as poly(D-lactic acid), poly(L-lactic acid) (called PLA), poly(glycolic acid) (called PLG), or else a mixture of poly( ⁇ -hydroxylated acids), such as a mixture of poly(D- and L-lactic acids), a mixture of poly(L-lactic acid) and of poly(glycolic acid), a mixture of poly(D-lactic acid) and of poly(glycolic acid), or a mixture of poly(D-lactic and L-lactic acids) and of poly(glycolic acid), which constitutes an embodiment of the invention.
  • the proportion of each constituent can be readily determined by those skilled in the art.
  • the preparation of the microparticles of the invention from at least one bioresorbable polymer can be carried out by any methods for preparing microparticles known to those skilled in the art, for which no stabilizer and no surfactant are used. In fact, step (i) of the method of the invention is characterized in that such agents are not used.
  • the stabilizers normally used in methods for preparing microparticles include, for example, poly(vinyl alcohol), pluronics (copolymer of poly(ethylene oxide) and of poly(propylene oxide)), and cationic or anionic surfactants such as cetyltrimethylammonium bromide or sodium dodecyl sulfate.
  • the dialysis for preparing the microparticles of the invention can be carried out with a solution of bioresorbable polymer in a water-miscible solvent, such as acetone, DMSO or DMF, at a concentration by mass of 0.1% to 10%, dialyzed against 1000 times its volume of water for 12 hours.
  • a water-miscible solvent such as acetone, DMSO or DMF
  • the second step of the method of the invention consists in bonding protein substances to the microparticles obtained in the first step of the method, without using surfactant.
  • This step of bonding the protein substance to the surface of the microparticles has the characteristic that it is carried out without surfactant.
  • the microparticles of the invention exhibit a colloidal stability that provides a range of particle size suitable for use in immunization.
  • surfactant normally used for the bonding of protein substances to the surface of microparticles, reference may be made to the surfactants mentioned above.
  • the protein substances to be bonded to the surface of the microparticles obtained in step (i) of the method of the invention may be any protein substance against which it is sought to trigger an immune response.
  • immune response is intended to mean a cell-mediated response, a humoral response or both.
  • cell-mediated response is intended to mean a response mediated by T lymphocytes and/or other leukocytes. This response is reflected by the induction of a lytic activity by cytotoxic T lymphocytes and/or by cytokine production by suppressor CD8+ T lymphocytes or by helper T cells.
  • the term “humoral response” is intended to mean a response mediated by the antibody molecules secreted by B lymphocytes.
  • the protein substances that are suitable for the purposes of the invention may be of several origins, such as of viral or bacterial origin.
  • antigens and epitopes By way of example of such protein substances, mention may, for example, be made of antigens and epitopes or any protein substance having the role of an antigen after bonding to the microparticles.
  • the antigens are molecules capable of being recognized by an antibody, the synthesis of which they have induced via an immune response, and containing at least one epitope. This may be whole proteins or protein fragments having conserved the structure of interest.
  • the epitopes are peptides comprising between 3 and 15 and generally between 5 and 15 amino acids, having also conserved the structure of interest.
  • the suitable viruses are any viruses for which substances capable of an immune response are known.
  • herpesviruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV), papilloma viruses (HPV) and human immunodeficiency viruses (HIV), such as HIV-1 and HIV-2.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HPV papilloma viruses
  • HIV human immunodeficiency viruses
  • nucleic acid sequences of the viruses suitable for the purposes of the invention, and also the proteins encoded by said sequences, are largely known to those skilled in the art and are available, for example, in databases such as GenBank.
  • the HIV virus has genes which encode structural proteins of the virus.
  • the gag gene encodes the protein that forms the core of the virion, including the p24 antigen.
  • the pol gene encodes the enzymes responsible for reverse transcription (reverse transcriptase), for cleavage (protease) and for integration (integrase).
  • the env gene encodes the envelope glycoproteins. It contains six other genes (tat, rev, nef, vif, vpr and vpu (HIV-1) or vpx (HIV-2)) which encode proteins involved in regulating the expression of the genes of the virus (regulatory proteins).
  • the HIV genome also comprises the 5′ and 3′ LTRs (Long Terminal Repeats) which comprise regulatory elements involved in the expression of the genes of the virus.
  • the protein substance used in the method of the invention is an HIV virus antigen.
  • the HIV virus antigen is a regulatory protein or the p24 protein, the preferred regulatory proteins being the Tat, Rev or Nef protein.
  • the 5′ end of its genome corresponds to an untranslated region adjacent to the genes which encode the structural proteins, the nucleocapsid core protein, the two envelope glycoproteins, E1 and E2, and a small protein called p7.
  • the 5′ untranslated region and the core gene are relatively well conserved in the various genotypes.
  • the E1 and E2 envelope proteins are encoded by regions that are more variable from one isolate to another.
  • the p7 protein is an extremely hydrophobic protein which is thought to constitute an ion channel.
  • the 3′ end of the HCV genome contains the genes which encode the nonstructural proteins (NS2, NS3, NS4, NS5) and a 3′ noncoding region that has a well-conserved domain (Major M E, Feinstone S M, Hepatology, June 1997, 25(6):1527-1538).
  • the NS3 nonstructural protein of HCV is a 630 amino acid protein which comprises two distinct structural domains: an N-terminal domain, of 81 amino acids, which has an active serine protease activity involved in the maturation of the viral protein (domain called NS3 protease), and a C-terminal domain, of 549 amino acids, comprising a helicase activity associated with an NTPase activity which plays a role in the replication of the viral genome (domain called NS3 helicase).
  • This NS3 protein is relatively well-conserved among the various genotypes of the virus, such that this protein constitutes a “vaccine candidate” antigen of choice.
  • the protein substance of interest is an antigenic protein of HCV, preferably a nonstructural protein, more preferably the NS3 protein, and in particular the NS3 helicase protein being more preferred.
  • the protein substances suitable for the purposes of the invention can be obtained by the genetic engineering technique which comprises the steps of:
  • the protein substances of interest when they are small in size, can also be prepared by conventional peptide syntheses well known to those skilled in the art.
  • the bonding of the protein substances to the bioresorbable microparticles can be carried out by any method known to those skilled in the art.
  • bondsing include adsorption, covalent bonding and bonding via a polysaccharide polymer deposited at the surface of the microparticle, such as chitosan, the protein substance being bonded to the chitosan by adsorption.
  • the adsorption can be carried out, for example, by mixing the microparticles with the protein substances and incubating with agitation, for example at ambient temperature or at 37° C.
  • the covalent bonding of the protein substances to the surface of the microparticles can be carried out using the techniques and reagents known in the literature, as described, for example, in Bioconjugate Techniques, G. T. Hermanson, Academic Press, London, 1996 and Chemical Reagents for Protein Modification, R. L. Lundblad, Ed. CRC Press, 1991.
  • the bonding of the protein substances to the microparticles is carried by adsorption.
  • bioresorbable microparticles to which protein substances are bonded prepared according to the method of the invention, are devoid of stabilizer and of surfactant, such that they are novel and constitute another subject of the invention.
  • microparticles of the invention because of their ability to induce an immune response by virtue of the protein substance, and because of their lack of toxicity, are particularly suitable for the preparation of pharmaceutical compositions, in particular vaccines, that are useful in the treatment of pathologies associated with the protein substance bonded to the microparticles.
  • Another subject of the invention consists of the use of the bioresorbable microparticles of the invention, for preparing a medicinal product.
  • the medicinal product prepared with the microparticles of the invention is particularly useful for the inhibition, prevention or treatment of an infection caused by a virus, such as, for example, the HIV or HCV virus or any other known virus, which constitutes another embodiment of the invention.
  • a virus such as, for example, the HIV or HCV virus or any other known virus, which constitutes another embodiment of the invention.
  • the invention also relates to a pharmaceutical composition, in particular a vaccine, containing at least one microparticle of the invention and, where appropriate, a pharmaceutically acceptable excipient.
  • compositions of the invention are suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal, rectal, intraocular or intra-auricular administration, it being possible for said active ingredient to be administered in unit administration forms.
  • the unit administration forms may, for example, be tablets, gelatin capsules, granules, powders, injectable oral solutions or suspensions, transdermal patches, sublingual, buccal, intratracheal, intraocular, intranasal or intra-auricular administration forms, forms of administration by inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous administration forms, rectal administration forms, or implants.
  • topical administration creams, gels, ointments, lotions or eye lotions can be envisioned.
  • Said unit forms contain a dosage so as to allow daily administration of from 0.001 to 10 mg of active ingredient per kg of body weight, according to the pharmaceutical form.
  • the dosage appropriate for each patient is determined by the physician according to the method of administration and the weight and response of the patient.
  • the present invention also relates to a method of treating pathologies associated with the protein substance bonded to the microparticle of the invention, which comprises the administration, to a patient, of an effective dose of a pharmaceutical composition of the invention.
  • microparticles of the invention also make it possible to obtain antibodies, which constitutes another subject of the invention.
  • the antibodies according to the invention are polyclonal or monoclonal antibodies, monoclonal antibodies being preferred.
  • polyclonal antibodies can be obtained by immunization of an animal with at least one microparticle of the invention, followed by recovery of the desired antibodies in purified form, by taking a sample of the serum of said animal and separating said antibodies from the other constituents of the serum, in particular by affinity chromatography on a column to which is attached an antigen specifically recognized by the antibodies, in particular that of the microparticle of the invention.
  • the monoclonal antibodies can be obtained by means of the hybridoma technique, the general principle of which is recalled hereinafter.
  • an animal is immunized with at least one microparticle of the invention, for which the B lymphocytes are then capable of producing antibodies against the protein substance of said microparticle.
  • These antibody-producing lymphocytes are then fused with “immortal” (murine in the example) myeloma cells so as to give hybridomas.
  • “immortal” murine in the example myeloma cells
  • Each hybridoma is multiplied in the form of a clone, each resulting in the production of a monoclonal antibody whose recognition properties with respect to the microparticle of the invention may be tested, for example, by ELISA, by one- or two-dimensional immunoblotting, by immunofluorescence, or using a biosensor.
  • the monoclonal antibodies thus selected are subsequently purified, in particular according to the affinity chromatography technique described above.
  • microparticles and the antibodies of the invention are also useful in the diagnosis of the pathological state associated with the protein substance bonded to the surface of said microparticles.
  • the microparticles or antibodies of the invention can be used as a partner for the capture or detection of an analyte in any diagnostic technique using such partners, such as the ELISA method.
  • any diagnostic technique using such partners such as the ELISA method.
  • an antibody of the invention obtained from microparticles to which said antigen is bonded is used, whereas, if the intention is to search for antibodies, the microparticles of the invention are used. In the latter case, if the diagnostic test requires the use of a solid support, the microparticles may or may not play this role.
  • Another subject of the invention consists of a diagnostic composition consisting of the bioresorbable microparticles or of the antibodies of the invention.
  • this diagnostic composition for the in vitro diagnosis of the pathological state related to the protein substance bonded to the bioresorbable microparticle, it being possible for the pathological state to be, according to one embodiment, a viral infection, as caused by the HIV virus or the HCV virus.
  • FIG. 1 gives the result of CTL assays by immunizing mice with the DNA sequence corresponding to the HCV NS3NS4 polyprotein as a control ( FIG. 1A ), with the NS3 helicase protein substance and Freund's adjuvant, without microparticle ( FIG. 1B ), with the NS3 helicase protein substance without microparticle ( FIG. 1C ), with PLA microparticles without protein substance ( FIG. 1D ), and with the microparticles of the invention in which the polymer is PLA and the protein substance is the NS3 helicase peptide ( FIG. 1E ),
  • FIG. 2 represents a histogram giving the anti-p24 IgG titers according to an ELISA assay, in the preimmune sera and the immune sera of six rabbits (L1 to L6) having been given an injection of the particles of the invention PLA-p24 either intradermally (ID) or subcutaneously (SC),
  • FIG. 3 represents histograms (mean of four replicates+/ ⁇ standard deviations) obtained according to an ELISPOT assay in two macaques M1 and M2 having received microparticles of the invention PLA/p24, giving the number of spots per million cells obtained as a function of the days post-immunization after stimulation without antigen (medium, negative control, black histogram), after stimulation with p24 (gray histogram) or after stimulation with the PMA-ionomycin couple (outline histogram),
  • FIG. 4 represents histograms giving the number of spots obtained by ELISPOT in two macaques M1 and M2 having received microparticles of the invention PLA/p24, after stimulation either with the p24 protein (black histogram), or with the peptides (gray histogram), in the total PBMC fraction, the total PBMC fraction in the presence of the anti-CD4 antibody, the CD4 + -depleted PBMC fraction (CD4 ⁇ PBMC) and the corresponding CD4 + -enriched fraction (CD4+ PBMC), and also the CD8 + -depleted PBMC fraction (CD8 ⁇ PBMC) and the corresponding CD8 + -enriched fraction (CD8+ PBMC),
  • FIG. 5 represents histograms giving the anti-p24 IgG titer obtained by ELISA in the M2 monkey having received microparticles of the invention PLA/p24, as a function of the days on which the sequential sera were taken,
  • FIG. 6 represents histograms giving the OD value obtained by ELISA in the preimmune serum, in the serum after the 1st immunization, in the serum after the second immunization and in the serum after the 3rd immunization of mice immunized with SRDC cells and sensitized either with the negative control (Ct1 and Ct2), or with the p24 protein (p24-1 and p24-2), or with the microparticles negative control (NanoOva1 and NanoOva2) or with the microparticles of the invention (Nanop24-1 and Nanop24-2),
  • FIG. 7 represents graphs giving the relative proliferative index (RPI) specific to the NS3h protein as a function of the amount of NS3h used in mice having received NS3h-PBS (PBS), of the microparticles of the invention prepared by dialysis (DYS), of the microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alum composition,
  • RPI relative proliferative index
  • FIG. 8 represents graphs giving the relative proliferative index (RPI) specific to the NS3h protein as a function of the amount of NS3h used in mice having received NS3h-PBS (PBS), of the microparticles of the invention prepared by dialysis (DYS), of the microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alum composition (Alum) in the cells of the popliteal nodes for the localized cellular response ( FIG. 8A ) and in the cells of the spleen for the systemic cellular response ( FIG. 8B ), and
  • RPI relative proliferative index
  • FIG. 9 represents histograms giving the cellular proliferative index (RPI) as a function of the immunogens used in mice, i.e. NS3h-PBS (PBS), of the microparticles of the invention prepared by dialysis (DYS), of the microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alu composition (Alum), in the cells of the spleen without stimulation (0), after stimulation with NS3h protein (1) or after stimulation with protein and the anti-CD4+ antibody (1+aCD4).
  • RPI cellular proliferative index
  • PLA 50 50% of poly(L-lactic acid) and 50% of poly(D-lactic acid)
  • molar mass 52,000 Da Phusis®
  • This PLA was dissolved at 2% by total weight of solution in DMSO (Prolabo®).
  • the organic solution of PLA was subsequently introduced into a dialysis membrane with a cut off of 15 000 Da (Spectrum®) and the assembly was placed in a water bath of double-distilled water (4 l, MilliQ®), stirred and changed regularly every hour, for 6 h. The final dialysis bath was continued overnight in order to obtain particles as a precipitate.
  • the particles thus obtained were characterized in terms of their size, their polydispersity index and their charge using the Zetasizer 3000 HS device (Malvern® Instruments). Their solids content was also evaluated after weighing, by means of the calculation: (mass of dry extract/mass of wet extract) ⁇ 100.
  • HIV-1 p24 protein was prepared in recombinant form in E. coli and was purified by metal-chelate affinity chromatography according to the technique of Cheynet V., et al., 1993, Protein Expr. Purif., 4:367-372.
  • the PLA microparticles were prepared as described in point 1 above and have a particle diameter of 515.7+/ ⁇ 6.7 nm, a solids content of 1.1% and a polydispersity index of 0.242+/ ⁇ 0.013.
  • HIV-1 Tat protein of sequence SEQ ID No. 1, synthesized according to the procedure described in Péloponippoe J. P., et al., 1999, The Journal of Biological Chemistry, 274(17):11473-11478, was used.
  • the PLA microparticles were prepared as described in point 1 above and have a particle diameter of 420.1+/ ⁇ 10.7 nm, a solids content of 1.02% and a polydispersity index of 0.241+/ ⁇ 0.040.
  • the gene encoding amino acids 1192-1458 corresponding to the helicase domain of the HCV NS3 protein as a fusion with hexahistidine was cloned into the prokaryotic expression vector pMH80 and expressed in E. coli JM109 bacteria (Promega).
  • the expression of the recombinant protein was carried out at 30° C. after 3 hours of induction with 1 mM IPTG (isopropyl-beta-D-thiogalactopyranoside, Promega).
  • the bacteria were lysed by sonication in the buffer solution: 10 mM Tris-HCl, pH 8, 5 mM MgCl 2 , 1% Triton X100, 1 tablet of anti-protease (Boehringer), 250U benzonase (Merck).
  • the soluble fraction was purified on a Ni-agarose column and eluted in 10 mM sodium phosphate buffer solution, pH 7.2, containing 300 mM NaCl and 300 mM imidazole. The pure protein was thus dialyzed against PBS, pH 7.2.
  • the protein was analyzed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), and mass spectrometry.
  • SDS sodium dodecyl sulfate
  • the degree of purity of the NS3 protein, helicase domain is estimated at greater than 95%.
  • the absence of endotoxin was verified by measuring the degree of endotoxins (LPS) with an in vitro LAL and functional assay.
  • the PLA microparticles were prepared as described in point 1 above and have a particle diameter of the order of 600 nm, a solids content of 1% and a polydispersity index of 0.2.
  • PLA 50 (50% of poly(L-lactic acid) and 50% of poly(D-lactic acid)) of molar mass 52 000 Da (Phusis®) was used.
  • This PLA was dissolved at 2% by a total weight of solution in acetone. The solution of PLA in acetone was then added, dropwise, to 35 ml of water and the solvent was evaporated off under reduced pressure for 35 min.
  • the particles thus obtained were characterized in terms of their size, their polydispersity index and their charge using the Zetasizer 3000 HS device (Malvern® Instruments). Their solids content was also evaluated after weighing, by means of the calculation: (mass of dry extract/mass of wet extract) ⁇ 100.
  • HCV NS3 helicase peptide of sequence SEQ ID No. 2 obtained in recombinant form as indicated in example 1, point 4 above was used.
  • the PLA microparticles were prepared as described in point 1 above and have a particle diameter of the order of 250 nm, a solids content of 1% and a polydispersity index of 0.3.
  • NS3 helicase peptide diluted to 0.327 g/l in a PBS buffer (Phosphate Buffered Saline buffer; 150 mM NaCl, pH 7.1), prepared as described in point 1 above, except that 6.8 ml of 0.1M PBS buffer, pH 9.2, were used, were mixed with 200 ⁇ l of the microparticles, and stirring was carried out overnight on a wheel at ambient temperature.
  • PBS buffer Phosphate Buffered Saline buffer; 150 mM NaCl, pH 7.1
  • mice 6 to 8 weeks old at the time of the first immunization.
  • mice received three successive doses (40 ⁇ g or 10 ⁇ g each) of the immunogens described in point 2 above at 0, 2 and 4 weeks. All the injections were given subcutaneously.
  • the animals were sacrificed 10 days (D38), 14 days (D42) or else 42 days (D70) after the third injection and the blood and the spleen were taken for the immunological analyses.
  • the humoral response and the cellular response were investigated as follows:
  • CTL Assay The cell suspension was placed in culture in the presence of a 9-mer peptide (AMQMLKETI, SEQ ID No. 3) which corresponds to an immunodominant H-2K d -restricted CTL epitope, and of IL-2. Five days later, the effector population was restimulated with irradiated na ⁇ ve cells loaded with the peptide. The effector cytotoxic population was harvested after the 7th day and the CTL activity was measured using 51 Cr-labeled P815 cells as targets.
  • AQMLKETI 9-mer peptide
  • ELISPOT makes it possible to determine the number of cells secreting a given cytokine in response to a specific stimulus.
  • the cell suspensions obtained from the spleens were restimulated in vitro with the peptide AMQMLKETI for 20 h in order to analyze the CD8-type responses.
  • 96-well ELISPOT plates with PVDF membranes Multiscreen IP, Millipore
  • an anti-IFN- ⁇ antibody The splenocyte suspensions were incubated in these plates so as to capture the cytokines secreted by each cell. The spots corresponding to each cell secreting the cytokine of interest were visualized with a biotinylated detection antibody specific for the cytokine of interest.
  • (iii) Proliferation The splenocytes were stimulated in the presence of the p24 protein for 5 days. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation. Following the pulse, the cells were harvested on a membrane which retains the DNA and makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the immunogen (p24).
  • mice 5 mice per branch
  • three doses of 10 ⁇ g of immunogen were administered to mice at D38
  • investigation of the humoral response and the CTL assay and proliferation as cellular response was carried out with 15 mice (5 mice per branch), three doses of 10 ⁇ g of immunogen and sacrifice of the mice at D38, and investigation of the humoral response and the CTL assay and proliferation as cellular response.
  • mice 3 or 4 mice per branch
  • the immunogens were used at a rate of 40 ⁇ g and that the mice were sacrificed either at D42 (3 mice) or at D70 (4 mice).
  • mice received three successive doses of the immunogens described in point 2 above, at 0, 2 and 4 weeks, at a rate of 50 ⁇ g each in the case of the proteins or of 100 ⁇ g each in the case of the naked DNA. All the injections were given subcutaneously, with the exception of the naked DNA, which was administered intramuscularly.
  • the animals were sacrificed approximately 70 days (D70) after the first injection and the blood and the spleen were taken for the immunological analyses.
  • the CTL cellular response was investigated as follows: after sacrifice of the mice, the spleens were removed sterilely in order to prepare a cell suspension. The cell suspension was placed in culture in the presence of the KLV peptide (KLVALGVNAV, SEQ ID No. 11), which corresponds to a CTL epitope contained in the NS3 protein, and of IL-2. Five days later, the effector population was restimulated with irradiated na ⁇ ve cells loaded with the peptide. The effector cytotoxic population was harvested after the 7th day and the CTL activity was measured using 51 Cr-labeled P815 cells as targets.
  • KLV peptide KLV peptide
  • FIG. 1 representing graphs giving the percentage specific lysis as a function of the effector/target ratio
  • FIG. 1A gives the cellular response induced after injection of the DNA sequence corresponding to the HCV NS3NS4 polyprotein as a control
  • FIG. 1B gives the cellular response induced after injection of the NS3 helicase/Freund's adjuvant combination without microparticle
  • FIG. 1C gives the cellular response induced after injection of the NS3 helicase peptide without microparticle
  • FIG. 1D gives the cellular response induced after injection of the PLA microparticles without protein substance
  • FIG. 1E gives the cellular response induced after injection of the PLA/NS3 helicase microparticles of the invention.
  • PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 above, and also the p24-Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion, and which is known to exhibit a good immunogenic capacity (positive control), were used.
  • the rabbits received five successive doses of 200 ⁇ g of the immunogens described in point 2 above at 0, 1, 2, 3 and 4 months. All the injections were given subcutaneously or intradermally.
  • FIG. 2 gives the anti-p24 IgG titers in the preimmune sera and the immune sera of the 6 rabbits (L1 to L6) having been given an injection either intradermally (ID) or subcutaneously (SC).
  • the titer corresponds to the inverse of the dilution for which an OD approximately equal to 0.1 is obtained.
  • the preimmune serum was taken at D0, before the injection of the immunogens, and the immune serum was taken at D0 plus 4 months.
  • the results obtained show that the immunization with the PLA/p24 particles of the invention gives good titers in all the animals, irrespective of whether the antigen was administered subcutaneously or intradermally. However, in the model chosen, intradermal administration appears to be slightly better than subcutaneous administration. It may be noted that the titers obtained with the immunization of Freund's/p24 are substantially comparable to those obtained with a PLA/p24 immunization (2 ⁇ 10 7 versus 5 ⁇ 10 6 ). The PLA/p24 microparticles can therefore be used to induce a polyclonal serum in rabbits.
  • the immunization experiments were carried out on cynomolgus macaques housed at the CEA.
  • This procedure makes it possible to determine the number of cells secreting interferon gamma (IFN-gamma) in response to an antigenic stimulation at a final concentration of 5 ⁇ g/ml for 48 h.
  • This procedure was used successfully with freshly isolated peripheral blood mononuclear cells (PBMCs), PBMCs cryoconserved beforehand, T lymphocytes lines derived from PBMCs stimulated in vitro and from PBMCs pre-depleted of CD4 + cells (using an anti-CD4 antibody) or of CD8 + cells (using an anti-CD8 antibody).
  • the CD4 + or CD8 + cell sorting was carried out using the MACS reagents, CD4 microbeads (Cat no. 130-091-102) and CD8 microbeads kit (Cat no. 130-091-112) from Miltenyi Biotec, according to the manufacturer's instructions.
  • the PMA-ionomycin couple (PMA for Phorbol Myristate Acetate), which mimics the effect of an antigenic activation of T lymphocytes, was used as a positive control.
  • 96-well ELISPOT plates with PVDF membranes Multiscreen, Millipore
  • PVDF membranes Multiscreen, Millipore
  • clone GZ-4 Mabtech, ref: 3420M-3
  • PBMCs were isolated from the blood samples on a Ficoll gradient, according to the usual techniques. 10 5 cells in 100 ⁇ l of culture medium/well and the antigen source in 100 ⁇ l of culture medium/well were deposited.
  • the antigen source is either the p24 protein, or a pool of gag peptides as defined hereinafter, for which it was verified beforehand that they make it possible to obtain a positive response in ELISPOT.
  • 4 ⁇ 10 3 cells in 200 ⁇ l of culture medium were deposited/well containing 50 ng/ml of PMA and 500 ng/ml of ionomycin. The plates were then incubated for 24 h at 37° C. in a humid atmosphere at 5% CO 2 , and then washed with PBS. The remaining cells were then lysed using a treatment with ice-cold water for 10 minutes, and the plates were again washed.
  • the visualizing antibody the biotinylated monoclonal directed against human IFN-gamma, clone 7-B6-1 (Mabtech, ref: 3420-6) was then added at 0.1 ⁇ g/well (incubation for 2 h at 37° C. or overnight at 4° C.). The spots were visualized by adding extravidin-alkaline phosphatase and the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate.
  • the sequences of the gag peptides used are as follows: EQIGWMTNNPPIPVG (SEQ ID No. 12) WMTNNPPIPVGEIYK (SEQ ID No.
  • NPPIPVGEIYKRWII SEQ ID No. 14
  • PVGEIYKRWIILGLN SEQ ID No. 15
  • IYKRWIILGLNKIVR SEQ ID No. 16
  • WIILGLNKIVRMYSP SEQ ID No. 17
  • GLNKIVRMYSPTSIL SEQ ID No. 18
  • IVRMYSPTSILDIRQ SEQ ID No. 19
  • YSPTSILDIRQGPKE SEQ ID No. 20
  • SILDIRQGPKEPFRD SEQ ID No. 21
  • IRQGPKEPFRDYVDR SEQ ID No. 22
  • PKEPFRDYVDRFYKT SEQ ID No.
  • FIG. 3 represents histograms (mean of 4 replicates+/ ⁇ standard deviations) giving the number of spots per million cells, obtained as a function of the days post-immunization, after stimulation without antigen (medium, negative control, solid histogram), after stimulation with p24 (hashed histogram) or after stimulation with the PMA-ionomycin couple.
  • the arrows under each graph represent the moment at which the injections were given (D0 and D+6 weeks).
  • ELISPOT responses make it possible to demonstrate what types of effector immune cells, CD4+ or CD8+, are responsible for the IFN-gamma secretion measured.
  • FIG. 4 represents histograms giving the number of spots obtained by ELISPOT after stimulation either with the p24 protein (solid histograms), or with the peptides (hashed histograms), in the total PBMC fraction, the total PBMC fraction in the presence of the anti-CD4 antibody, the CD4 + -depleted PBMC fraction (CD4 ⁇ PBMC) and the corresponding CD4 + -enriched fraction (CD4+ PBMC), and also the CD8 + -depleted PBMC fraction (CD8 ⁇ PBMC) and the corresponding CD8 + -enriched fraction (CD8+ PBMC). It should be noted that the amount of cells obtained in the M1 monkey was not sufficient to carry out the depletion experiments with the anti-CD8 antibody.
  • the results obtained in FIG. 4 show that the secretion of the IFN-gamma cytokine is carried out by both the CD4 ⁇ fraction and the CD8 ⁇ fraction of the peripheral blood mononuclear cells. Furthermore, there is no ELISPOT response in the presence of an anti-CD4 antibody or of an anti-CD8 antibody (positive sorting fractions), indicating that each of these antibodies interferes with the immune response. This experiment therefore makes it possible to conclude that the IFN-gamma secretion observed is mediated by both CD4+ effector cells and CD8+ effector cells. Furthermore, this observation is valid irrespective of the nature of the antigenic stimulation used, p24 protein or pool of gag peptides. It is important to underline that this is the first time that a microparticle-based formulation has made it possible to induce, in primates, a CD8+ response specific for the antigen of interest.
  • FIG. 5 represents histograms giving the anti-p24 IgG titer in the M2 monkey as a function of the days on which the sequential sera were taken.
  • the SRDC (H-2 k ) murine spleen cell line was used in order to transport the various immunogens to be tested.
  • This dendritic cell line was sensitized for 12 h with one of the following immunogens: PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2 (at 10 ⁇ g/ml of p24/PLA in terms of p24 equivalent), PLA-OVA microparticles, prepared in the same way, which will serve as a negative control, or p24 protein.
  • mice were divided up into 4 batches of 6 animals. Each mouse received 5 ⁇ 10 5 sensitized SRDC cells at 0, 2 and 4 weeks, subcutaneously. The 4 batches of mice received either nonsensitized cells (negative control), or cells sensitized with the PLA/p24 microparticles or PLA/OVA microparticles (microparticle negative control) or the p24 protein.
  • FIG. 6 represents histograms that give the OD value obtained by ELISA in the preimmune serum, in the serum after the 1 st immunization, in the serum after the second immunization and in the serum after the 3 rd immunization, of the mice sensitized either with the negative control (Ct1 and Ct2), or with the p24 protein (p24-1 and p24-2), or with the microparticle negative control (NanoOva1 and NanoOva2) or with the microparticles of the invention (Nanop24-1 and Nanop24-2).
  • the T lymphocytes isolated from the spleen and from the mucosal lymph nodes were placed in culture for 5 days in complete culture medium in the presence of various concentrations of the antigen of interest, and then radiolabeled with 3 H thymidine for 18 h.
  • the level of thymidine incorporation corresponds to the degree of the lymphoproliferative response.
  • the T lymphocytes isolated from the spleen and from the mucosal lymph nodes were placed in culture for 3 days in complete culture medium in the presence of various concentrations of the antigen of interest, and then the cytokine secreted into the culture supernatant were assayed using commercial ELISA kits.
  • mice having developed an antibody (IgG) response Lymphoproliferation Groups of after the 3 rd Stimulation index IFN-gamma mice injection Spleen-lymph node secretion Control 0/2 1 - 1 No SRDC-p24 0/2 1 - 1 No SRDC- 0/2 1.4 - 1.4 No PLA-Ova SRDC- 2/2 2.8 - 2.2 +spleen, PLA-p24 ++lymph node
  • the results show that the SRDC-PLA-p24 group is the only group for which it is possible to demonstrate a specific immune response.
  • the stimulation index is 2.8 for the cells isolated from the spleen and 2.2 for those isolated from the mesenteric lymph nodes.
  • the SRDC-PLA-p24 group is the only group which makes it possible to induce secretion of IFN-gamma, which is a Th1-type cytokine. This secretion in the presence of the antigen can be demonstrated using both the spleen cells and those of the mesenteric lymph nodes.
  • PLA-p24 microparticles in order to sensitize dendritic cells with an antigen of interest, in this case p24.
  • administration of the SRDCs when they are loaded with the PLA-p24s, makes it possible to induce both cellular and humoral specific responses, the SRDCs sensitized with the soluble p24 do not make it possible to induce anti-p24 responses.
  • the PLA microparticles can also be used successfully in immunotherapy applications based on the transfer of dendritic cells sensitized in vitro.
  • the PLA microparticles carrying the antigen can be used to facilitate its uptake by the dendritic cells.
  • the use of the microparticles of the invention makes it possible to considerably enhance the specific immune responses obtained.
  • mice 6 to 8 weeks old at the time of the first immunization.
  • mice per immunization group were used: the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • PADYS NS3 helicase genotype 1b protein
  • PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2 above
  • NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • mice received one dose (100 ⁇ g) of the immunogens described in point 2 above, subcutaneously into the plantar footpad.
  • the animals were sacrificed 10 days after the first injection and the popliteal lymph nodes were removed for immunological analysis.
  • the early and localized NS3h protein-specific dose-response cellular response was investigated as follows: Proliferation of the popliteal lymph node cells after stimulation with various concentrations of the NS3h protein—the popliteal lymph node cells were stimulated in the presence of 0, 0.1, 0.3 and 1 ⁇ M of the NS3h protein for 3 days. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation.
  • the cells were harvested on a membrane which retains the DNA and makes it possible to eliminate the nonincorporated labeled thymidine by washing.
  • FIG. 7 representing the graphs giving the relative proliferation index (RPI) specific to the NS3h protein, corresponding to the ratio of cpm (counts per minute) obtained for each concentration of NS3h (0, 0.1, 0.3 and 1 ⁇ M) relative to the zero concentration of the NS3h protein, as a function of the amount of NS3h used for the restimulation for the cell proliferation assay, in mice having received NS3h-PBS (PBS), microparticles of the invention prepared by dialysis (DYS), microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alum composition.
  • RPI relative proliferation index
  • This graph shows a cellular response specific for the NS3h protein when the PLA/NS3h microparticles prepared by solvent displacement (DDS) and the PLA/NS3h microparticles prepared by dialysis (DYS) are injected.
  • the specific cellular responses of the mice injected with the PLADDS/NS3hs of the invention are greater than those obtained with the PLADYS/NS3hs of the invention, the positive control (Alum/NS3h) and the PBS/NS3h control for all the concentrations of NS3h antigens tested (PLADDS/NS3h>PLADYS/NS3h>Alum/NS3h PBS/NS3h).
  • results show that enhanced cellular proliferative responses are obtained with the PLADDS/NS3h and PLADYS/NS3h microparticles of the invention, compared with the NS3h protein alone or adjuvanted with Alum.
  • mice 6 to 8 weeks old at the time of the first immunization.
  • mice per immunization group were used: the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • PADYS NS3 helicase genotype 1b protein
  • PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2 above
  • NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • mice received 2 doses (100 ⁇ g) of the immunogens described in point 2 above, the first dose having been given subcutaneously into the plantar footpad at day 0 and the second subcutaneously at the base of the tail at day 7.
  • the animals were sacrificed 7 days after the second injection and the popliteal lymph nodes and the spleen were removed for immunological analysis.
  • the NS3h protein-specific dose-response cellular response localized in the popliteal lymph nodes and systemic in the spleen was investigated as follows:
  • Proliferation of the popliteal lymph node cells after stimulation with various concentrations of the NS3h protein the cells of the popliteal lymph nodes and of the spleen were stimulated in the presence of 0, 0.1, 0.3 and 1 ⁇ M of the NS3h protein for 3 days. After 3 days of culture, 50 ⁇ l of supernatant were removed. The cells were pulsed for 18 h with tritiated thymidine, which incorporates into the DNA of the cells undergoing proliferation. Following the pulse, the cells were harvested on the membrane that retains the DNA and that makes it possible to eliminate the nonincorporated labeled thymidine by washing. The more the cells proliferate in response to the specific stimulus, the more the DNA is labeled; in other words, the greater the cellular response against the NS3h immunogen.
  • FIG. 8 representing the graphs giving the relative proliferation index (RPI) specific to the NS3h protein as a function of the amount of NS3h used (in ⁇ M) in mice having received NS3h-PBS (PBS), microparticles of the invention prepared by dialysis (DYS), microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alum composition (Alum), in the cells of the popliteal lymph nodes for the localized cellular response ( FIG. 8A ) and in the cells of the spleen for the systemic cellular response ( FIG. 8B ).
  • RPI relative proliferation index
  • FIG. 9 represents histograms giving the cellular proliferation index (RPI) as a function of the immunogens used in mice, i.e. NS3h-PBS (PBS), microparticles of the invention prepared by dialysis (DYS), microparticles of the invention prepared by solvent displacement (DDS) and the NS3h-Alum composition (Alum), in the spleen cells without stimulation (0), after stimulation with NS3h protein (1) or after stimulation with protein and anti-CD4+ antibody (1+aCD4).
  • RPI cellular proliferation index
  • interferon gamma IFN-gamma
  • mice 6 to 8 weeks old at the time of the first immunization.
  • mice per immunization group were used, the NS3 helicase genotype 1b protein (NS3h) alone, the PLA/NS3h microparticles of the invention prepared by dialysis (PLADYS) as indicated in example 1, point 4 above, the PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2, point 2 above, and also the NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • PLA/NS3h microparticles of the invention prepared by dialysis
  • PLA/NS3h microparticles of the invention prepared by solvent displacement (PLADDS) as indicated in example 2 above
  • NS3h-Alum (Pierce) composition prepared in the form of an emulsion and known to be an adjuvant in commercial vaccines (positive control).
  • mice received 3 doses (50 ⁇ g) of the immunogens described in point 2 above, subcutaneously at the base of the tail at 0, 2 and 4 weeks.
  • the sera were taken at day 13, day 27 and day 45 for the analyses of the specific humoral response against the NS3h protein using an ELISA assay as indicated hereinafter.
  • the animals were sacrificed at 10 days after the first injection and the popliteal lymph nodes were removed for immunological analysis.
  • the immunized mice sera were serially diluted.
  • the reaction was visualized using peroxidase-labeled goat anti-mouse IgG1 serum (Southern Biotechnology Associates Inc., Cat no. 1070-05, Birmingham, Ala., USA), and a peroxidase-labeled goat anti-mouse IgG2a antibody (Interchim, UPB 90520).
  • the IgG2a/IgG1 isotype ratio which makes it possible to interpret the IFN-gamma/IL-4 (respectively, Th1-Th2) tendency of the immune response was also determined.
  • PLA/NS3h microparticles makes it possible to obtain an essentially IgG1 antibody response specific for the NS3h protein, suggesting that the response is of the Th2 type.
  • This study consists in studying the effect on antigen-presenting cells of the NS3h protein adsorbed onto PLA nanoparticles. To do this, the procedure is carried out in the presence of dendritic cells generated using monocytes isolated from human peripheral blood and differentiated.
  • the ability of the potential adjuvant and of the PLA-NS3h formulation to promote the differentiation and maturation of monocytes into dendritic cells is also tested. This study enables us to understand more clearly their role in cell mediation, which is essential at the interface between innate and adaptive immunity.
  • the screening is carried out by means of successive steps of analysis of phenotypic markers of differentiation and of maturation and by analysis of the profile of cytokines produced. The analysis of the cytokine production makes it possible to identify whether the PLA-NS3 formulation induces a Th1 and/or Th2 profile.
  • the monocytes were isolated from normal human peripheral blood (recovered at the Etableau Francais du Sang [French bloodbank] in Lyon) by centrifugation on a Ficoll and Percoll (Amersham Biosciences) gradient.
  • the Ficoll makes it possible to create a density gradient while at the same time conserving the integrity of the cells and their function.
  • the red blood cells and the polymorphonuclear cells that are more dense than the Ficoll are at the bottom of the tube.
  • the PBMCs peripheral blood mononuclear cells
  • lymphocytes and monocytes remain at the interface between the plasma and the Ficoll. They are then purified on a Percoll gradient. After centrifugation, the lymphocytes, that are more dense than the Percoll, are in the pellet, whereas the monocytes remain at the interface between the medium and the Percoll.
  • the monocytes were incubated with the mixture of antibodies (Ab) indicated hereinafter, in order to eliminate the remaining contaminants by depletion of T lymphocytes (mouse anti-CD3 Ab OKT3, ATCC, Rockville, Md., USA, directed against T lymphocytes), of B lymphocytes (mouse anti-CD19 Ab hybridoma 4G7, directed against B lymphocytes), of red blood cells (mouse anti-glycophorin A Ab, Immunotech) and of NK cells (mouse anti-CD56 Ab NKH1, Immunotech, directed against Natural Killer cells) and using magnetic beads (Dynal).
  • the Dynal beads are small magnetic beads coated with sheep antibodies directed against mouse antibodies. These antibodies will bind the mouse Ab/cell complexes and then, after the cell suspension has been passed over a magnetized carrier, the remaining cells will only be monocytes.
  • the cell suspension was analyzed by isolated passage of the cells through a liquid matrix.
  • the passage of these cells through a light beam results in two types of scattering referred to as small angle (Forward scatter-FS) and large angle (Side scatter—SS), which represent the two cellular parameters taken into account, the size (scattering) and the granulometry (refraction) of the cells.
  • the use of antibodies coupled to fluorochromes FITC: Fluorescein IsoThioCyanate read on the X-axis FL1 and PE: PhycoErythrin read on the Y-axis in FL2) that are laser-excitable allows detectable fluorescence emission.
  • FITC Fluorescein IsoThioCyanate read on the X-axis FL1
  • PE PhycoErythrin read on the Y-axis in FL2
  • An electrical signal amplification and analog-digital converter system allows the data to be computer-formatted.
  • the contaminant level is less than 10%.
  • the monocytes were subsequently placed in culture (day 0) in 24-well plates at a rate of 1 ⁇ 10 6 cells/ml in RPMI 1640 medium (Gibco), 2 mM of L-glutamine (Life Technologies), 10 mM Hepes (Life Technologies), 40 ng/ml of gentamycin (Life Technologies)+10% decomplemented fetal calf serum, and in the presence of GM-CSF (40 ng/ml) (Granulocyte Macrophage Colony Stimulating Factor) and recombinant human IL-4 (250U/ml) The GM-CSF and the IL-4 allow differentiation of the monocytes into immature dendritic cells.
  • a dose-response was carried out for the NS3h alone, the particles alone and the PL/NS3h particles.
  • the formulations were brought into contact with the immature dendritic cells in order to judge the ability of the PLA/NS3h particles to generate mature dendritic cells (adjuvant effect).
  • the NS3h protein gives no positive labeling.
  • the PLA/NS3h particles give very advantageous results. Specifically, it can be noted that the degree of activation of maturation is substantial. Right from the assay at 10 ⁇ l (0.24 mg PLA/10 ⁇ g helicase), maturation can be observed.
  • the PLA/NS3hs added to DCis induce the expression of all the activation markers (CD83+, CD86+, CD80+, HLA-DR+, CD40+).
  • the assay at 100 ⁇ l was carried out in the knowledge that it is in large excess in the conditions used in vitro.
  • the PLA formulations have an adjuvant effect on the HCV NS3h protein since they make it possible to obtain mature dendritic cells.
  • mice 6 to 8 weeks old at the time of the first immunization.
  • PLA/p24 microparticles of the invention prepared as indicated in example 1, point 2, and also the p24-Freund's adjuvant (Sigma) composition prepared in the form of a water-in-oil emulsion, and which is known to have a good immunogenic capacity (positive control), were used.
  • mice received 3 successive doses of 10 ⁇ g of the immunogens described in point 2 above, at 0, 2 and 4 weeks. All the injections were given subcutaneously. At D68 after the first injection, the humoral responses were restimulated with an intravenous injection of 50 ⁇ g of p24.
  • a mouse of the PLA-p24 group was sacrificed; the blood and the spleen were taken.
  • the splenocytes obtained from the spleen were placed in culture with Sp2/0-Ag14 myeloma cells so that they would fuse and become immortalized, according to the protocol described by Köhler and Milstein (Köhler, G. and Milstein, C., 1975, Nature, 256:495-497; Köhler, G. and Milstein, C., 1976, Eur. J. Immunol., 6:511-519).
  • the supernatants of the hybridomas obtained were screened in order to determine the presence of anti-p24 antibodies using the ELISA assay described in point 4 of this example.
  • the positive hybridoma colonies were subcloned twice according to the limiting dilution technique.
  • the anti-p24 antibody titer in the serum of the mice was determined just before sacrifice, individually for each mouse.
  • the titers obtained are comparable in the two groups. As monoclonal antibodies had already been obtained by Freund's/p24 immunization, we sought to determine whether the PLA/p24 immunogen, which makes it possible to induce comparable titers, will also make it possible to obtain monoclonal antibodies.
  • a mouse of the PLA/p24 group (mouse 1) was sacrificed and the cells from its spleen were fused with myeloma cells.
  • the hybridomas derived from the fusion were cloned by limiting dilution in 18 96-well plates. Screening of the hybridoma culture supernatants using an anti-p24 ELISA assay made it possible to identify 12 hybridoma clones which secrete a p24-specific antibody.
  • the PLA/p24 microparticles can therefore also be used to obtain monoclonal antibodies.

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US20090123493A1 (en) * 2005-11-14 2009-05-14 Centre National De La Recherche Scientifique (Cnrs) Composition Comprising a Colloidal Synthetic Bioresorbable Vector and a Viral Vector
US20100136043A1 (en) * 2000-09-14 2010-06-03 Austin Research Institute Composition comprising immunogenic microparticles

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CN105004862B (zh) * 2015-07-06 2017-01-25 山东博科生物产业有限公司 一种高通量联合检测丙肝病毒抗原抗体的试剂
WO2017120483A1 (fr) * 2016-01-08 2017-07-13 Oregon Health & Science University Compositions de ligands de récepteurs de lymphocytes t recombinés et méthodes de traitement d'un cancer de la prostate
WO2020247874A2 (fr) * 2019-06-07 2020-12-10 Diomics Corporation Administration topique à action retard faisant appel à un biopolymère en couches

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FR2692147B1 (fr) * 1992-06-15 1995-02-24 Centre Nat Rech Scient Microsphères en polymère biorésorbable exemptes de tensioactif, leur préparation et leur application comme médicament.
GB9514285D0 (en) * 1995-07-13 1995-09-13 Univ Nottingham Polymeric lamellar substrate particles for drug delivery
WO1998033487A1 (fr) * 1997-01-30 1998-08-06 Chiron Corporation Utilisation de microparticules contenant un antigene adsorbe dans le but de stimuler les reponses immunitaires
ES2260923T3 (es) * 1998-07-29 2006-11-01 Chiron Corporation Micorparticulas con superficies adsorbentes, procedimientos de fabricacion y uso de las mismas.
CA2388676A1 (fr) * 1999-10-13 2001-04-19 Chiron Corporation Procede d'obtention de reponses immunes cellulaires de proteines
US7501134B2 (en) * 2002-02-20 2009-03-10 Novartis Vaccines And Diagnostics, Inc. Microparticles with adsorbed polypeptide-containing molecules
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US20100136043A1 (en) * 2000-09-14 2010-06-03 Austin Research Institute Composition comprising immunogenic microparticles
US8287877B2 (en) 2000-09-14 2012-10-16 PX Biosolutions Pty Ltd. Composition comprising immunogenic microparticles
US8846026B2 (en) 2000-09-14 2014-09-30 Px Biosolutions Pty Ltd Composition comprising immunogenic microparticles
US20090123493A1 (en) * 2005-11-14 2009-05-14 Centre National De La Recherche Scientifique (Cnrs) Composition Comprising a Colloidal Synthetic Bioresorbable Vector and a Viral Vector
US9433674B2 (en) 2005-11-14 2016-09-06 Centre Nationale De Recherche Scientifique Composition comprising a colloidal synthetic bioresorbable vector and a viral vector

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