WO2003015743A1 - Immunisation by means of the local implantation of antigens which are irreversibly fixed to a bioadherent - Google Patents

Abstract

The invention relates to immunisation by means of the local implantation of antigens which are irreversibly fixed to a bioadherent in such a way as to induce or stimulate an immune response in humans or animals. The inventive method consists in irreversibly fixing antigens to the support by means of covalent bonds and not adsorption. The antigens, which are permanently fixed to said support, retain their immunogenic character. In this way, dangerous or virulent molecules and organisms can be safely presented to the organism.

Description

Immunization by local implantation of antigens irreversibly fixed on a bioadherent.

The present invention relates to a new immunization method consisting in inducing or stimulating an immune reaction in humans or animals by inserting an immunogenic implant consisting of a chemical adherent for biological use, or bioadherent, on the surface of which antigens of biological origin (tissue, cells, microsomes, microorganisms, natural or synthetic biological molecules) or of chemical origin (simple molecules or in the form of complexes, chemical functional groups) are fixed irreversibly. The immunogenic implant thus formed is inserted either into a blood vessel, partially and temporarily, or into a tissue, partially or completely, temporarily or permanently.

By design, this method is different from the immunization methods developed to date. In fact, despite the many medical advances of the 20th century, infectious diseases remain the most important problem of global public health. As we know, when an immunogenic material is introduced into the body, the immune system responds by generating specific molecular and cellular protection against antigens.

Conventional vaccination methods consist in inoculating by subcutaneous injections, attenuated doses of germs to effectively stimulate, in some cases, immunity to certain pathogens. This has saved many lives threatened by infectious diseases.

These vaccines are generally solutions which are composed of antigens (living germs, attenuated or inactivated, purified or synthetic antigenic material, active viral vectors, antibodies, DNA ...) associated with an adjuvant which often has the role of increasing the immune response.

The disadvantage of these technologies is that they are done empirically because the response to an antigen is variable. They are sometimes poorly supported and very often require one or more reminders to maintain this response. In addition, the method of vaccination by injection of an immunogenic solution unfortunately finds its limits in many applications. Indeed, it is still difficult to attenuate certain virulent strains or to develop a vaccine for certain applications: fight against tropical diseases (example: malaria), avoid the appearance of cancer cells, development of a contraception, stimulation of mucosal immunity in general leading to secretion of antibodies in the mucus and particularly genital mucus to stop the transmission of sexually transmitted diseases (genital herpes, chlamydia infections, AIDS ...), etc. Attempts to vaccinate the intestinal mucosa by mouth, in the same way as with the polio vaccine, have been shown to be ineffective since antigens administered by this route must cross the acidic environment of the stomach to reach plaques. of Peyer's present in the intestines.

To remedy this problem, techniques consisting in incorporating antigens with viral or bacterial subunit in liposomes containing a surfactant to form a more active complex than the free antigen [Fullerton W. EP 0 017 557 A2] have been developed to enhance the body's immune response to these antigens.

This idea of attaching biological or pharmaceutical active agents to carrier particles of all forms is experiencing particular growth thanks to advances made in the field of biomaterials. The study of biomaterials is an emerging science offering, thanks to the development of new biocompatible materials, promising prospects in terms of implants.

Biomaterials designate artificial or synthetic biocompatible materials intended to work under biological constraints and in direct contact with the living system. They are used for the production of materials implanted in humans or in contact with their biological fluids. They generally have specific applications which depend on their chemical and mechanical properties. They are schematically classified into three categories: metals and alloys, ceramics, and polymers.

Biocompatibility is the adequacy between the implant and its insertion environment. Indeed, the material must not cause physiological disturbances and must not be damaged due to the physiological environment in which it is placed. The material and the products of its wear must not induce toxicity, either by themselves or by degradation or salting-out, or cause harmful reactions on the part of the host organism (carcinogenicity, immune or rejection reaction, thrombosis, etc.).

Among modern vaccination methods, technologies for encapsulating antigens from biodegradable polymer devices, which allow their controlled release, were developed in 1976 by LANGER and FOLKMAN [1, 2] (*).

(*): Text reference articles are indexed with a reference number on page 13 "Bibliographic references", with the exception of patents, this for the sake of clarity and better readability of the document by the reader. TICE T., et al. [WO 89/08449] have invented a method for potentiating an immunization response capable of delivering an antigen to an animal, and comprising the steps of encapsulating effective amounts of the bioactive agent in a biocompatible excipient to form microcapsules . These microcapsules (<10μm in diameter) consist of a bioactive polymer or copolymer capable of passing through the gastrointestinal tract without any degradation or little degradation, so that the bioactive agent reaches Peyer's plaques or other associated lymphoid tissues to the mucosa in an effective amount to stimulate the systemic or mucosal immune system. Other inventors, ANDRIANON A. et al. [WO 95/02416], have developed a method of microencapsulated vaccine based on a hydrogel. This material consisting of alginate and polyphosphazene is used to encapsulate antigens and form microparticles (15μm in diameter or less) which can be administered parenterally or through the mucous membranes. These microparticles by adhering to the mucosa of the digestive tract, thus increase the absorption of antigens. Systems composed of biodegradable polymers have been used for a prolonged action of release of antigens in controlled quantity. Thus, SUZUKI T. et al. [WO 98/07443] have imagined an oral vaccine based on microspheres having a multilayer structure which consists of a core layer containing the antigens, and of several layers of envelope to surround this core. The disadvantages of the encapsulation methods relate to the control of the kinetics of release of the antigen. Indeed these kinetics can be altered by modification of the preparation of these polymers [3], by variation of the percentage of the copolymers [4], or by change of the size of the microspheres [5]. In addition, when biodegradable polymers degrade in the body after releasing antigens in the body, they can act as an adjuvant themselves, and produce two distinct immune responses.

Among the other methods for inducing an immune response, there is that of implants consisting of a biomaterial impregnated with antigens. STERΝICK J. [O 85/03635], as well as other authors [6], obtain the induction of an immune response by the implantation of a spongy substrate impregnated with antigen in the peritoneal cavity of a mammal .

Carrier particles of pharmacological agents have been produced from 60 nm synthetic nuclei [7]. Besides, nanocrystalline or inorganic particles, covered with adsorbed antibodies are often used to locate or reveal cellular or molecular components in light or electron microscopy or in the context of immunoblots [8, 9].

KOSSONSKY et al. [US patents nos. 5,219,577 and US 5,178,882] have coated these particles with a very fine layer of sugar (cellobiose, nitrocellulose, etc.) or oligonucleotides (<5 nm), before adsorbing proteins there. viral to obtain artificial viruses and thus obtain an immune response without danger of infection.

Authors have observed, in vitro, an increase in the immune functions of T cells, coming from sick patients, and put in the presence of a suture on which are adsorbed antiCD3 / antiCD28 monoclonal antibodies [10]. Despite these advances, these methods present some risks, since the introduction of microcapsules is not without long-term danger. Although the risk is low, it is possible that they may accidentally enter traffic when inoculated. In addition, the fact of injecting or implanting in a place large doses of microcapsules can lead to various serious pathologies: occlusion of microvessels, creation of aggregates resulting in the formation of cysts, benign or even cancerous tumors [11, 12, 13, 14]

Up to now, no implantation process has made it possible to bring antigens into direct and safe contact with the circulating blood. There are also no methods which allow the implant to be easily removed, without recourse to the surgical act, once the immune reaction has taken place, whether at the level of a vessel or of a tissue. . There is also no method to remove the implant accompanied by all of its antigens, because either these are released in the body (in the case of conventional vaccination) or they can detach from the support by desorption. The method according to the invention overcomes these aspects. The present invention relates to a new immunization method consisting in inducing or stimulating an immune reaction in humans or animals by inserting an immunogenic implant consisting of a chemical adherent for biological use, or bioadherent, on the surface of which antigens of biological origin (tissue, cells, microsomes, microorganisms, natural or synthetic biological molecules) or of chemical origin (simple molecules or in the form of complexes, chemical functional groups) are fixed irreversibly. The immunogenic implant thus formed is inserted either into a blood vessel, partially and temporarily, or into a tissue, partially or completely, temporarily or permanently. The principle on which the process is based, consists in inducing an immune response, by introducing an immunogenic material which is not phagocytosed quickly by the cells of the host organism, contrary to the case of conventional vaccines or microbeads or vaccinating nanoparticles. To prevent antigens from disappearing too quickly in the body by phagocytosis or catabolism, these are fixed irreversibly on a non-circulating material, and immobilized within a vessel or tissue.

The antigens attached to the material attract a large number of immune cells, in particular neutrophilic granulocytes and phagocytes (monocytes and macrophages) active in the blood and tissues. These cells will phagocyte partly or entirely these antigens and trigger the specific immune system in which macrophages, humoral antibodies and different types of lymphocytes collaborate closely, ultimately leading to the production of antibodies. This process makes it possible to trap microorganisms, molecules, dangerous antigenic cells etc., to expose them to circulating immune cells without risk of infection for the organism.

For example, it is known that the dangerousness of virulent organisms, like the case of capsule viruses, comes from the fact that, when they are in free circulation, they adhere to the surface of certain target cells of the organism host through specific receptors, before fully entering the cytoplasm to spill and duplicate their genetic material. The present invention makes it possible to partially and definitively enclose part of the viral capsule in the adherent material, in order to present to the immune cells of the organism only the antigens present on the emerging part, accessible to antibodies, to phagocytic cells or complement factors. This landlocking on the adherent support therefore prevents the internalization of the virus at the level of non-phagocytic immune cells and thereby prevents infection of the host organism.

In addition, this process allows the insertion into the body of the material to obtain a lower catabolic rate for the degradation of antigens than current processes. A low catabolic rate helps maintain the immune response and prevents the need for subsequent boosters.

It is necessary that the connection established between the biological or chemical material and its support is irreversible, stable, and durable otherwise there is a risk to introduce into the body of dangerous organisms or molecules, especially in case of prolonged implantation, for the physico-chemical agents contained in the blood, by progressively degrading the binding of the antigen to its support, risk releasing infectious or toxic agents, and this well before a specific immune response is triggered. In the context of this process, the use of chemical adhesives for multiple biological or bioadherent uses have the best adhesion and biocompatibility characteristics for safe application. The adhesives are designed from polymers, polymer resin or silicone. The characteristic of the bioadherent is that it establishes chemical bonds with the antigens to be bound, during its polymerization. These chemical bonds are covalent bonds between the atoms of the antigen (example: amino, carboxyl or hydroxyl groups of the protein structure) and the functional groups of the bioadherent. These bonds, irreversible, are therefore stronger than in an adsorption or a simple mechanical interaction with a cement. The most used bioadherents and currently having the best adhesion characteristics are cyanoacrylates (akyl-α-cyanoacrylate, methyl- and ethyl-2-cyanocrylate, polyurethanes, cyanoacrylate 2-n-butyl, gelatin-resorcinol complex crosslinked by formaldehyde ...). Cyanoacrylates are the most tested and used ideal biological adherent. The use of bioadherents has developed in particular in numerous clinical applications, including aid for tissue repair [15, 16, 17, 18], and for suturing blood vessels [19, 20]. The suturing properties and therefore the adhesion to the tissues of certain cyanoacrylates sometimes prove to be superior to certain adhesives based on purely biological molecules [21]. Pharmaceutical applications involving micro or nanoparticles of non-bioadherent cyanoacrylates in the field of transport of active molecules, by adsorption, have been developed [22, 23]. If these micro or nanoparticles of cyanoacrylates increase the diffusion of physiological molecules [22], they have also recently served both as adjuvant and transporter of antigens [24, 25, 26,] or transporter of antibodies used as vector directed on a specific target [27]. Despite their interest, these methods involve certain non-negligible risks, all the more so since all of the techniques mentioned, from conventional vaccination to current methods using biomaterials, require one or more reminders. high, the use of micro or nanoparticles represents a risk which is difficult to take, especially since their exact role in diffusion and transport is unknown [22] as well as the effects of short repetitive injections or long term. The method according to the invention makes it possible to avoid these risks with two possible options: either by partially introducing the immunogenic implant into the body and removing it after use, without surgical intervention, or by leaving it in the body in a form (size and geometry) which does not allow its diffusion in the various tissues (pastilles, needles, flexible or rigid filaments, rods, films ...)

The mechanical properties of the material must be compatible with the environmental conditions imposed on it in its place of insertion (biophysical constraints, physico-chemical processes, etc.). The bioadhesive can be previously spread on a material of variable nature: carbon, organic compounds comprising: synthetic polymers or copolymers made from different families of polymers (polyolefins, vinyl, styrenics, acrylics, polyamides, saturated polyesters, polycarbonates, polyacetals , fluoropolymers, silicones, polyurethanes), silicones, natural biopolymers (example: cellulose, silk ...), modified natural biopolymers (example: polysaccharide with a carboxylic function ...), artificial biopolymers (e.g. homopolypeptide ...) or artificial biocopolymer (example: copolymer of lactic and glycolic acid), non-organic materials including appropriate metals (gold, silver, nickel, platinum ...) and alloys, ceramics, fabrics treated specifically for give them the essential biomechanical properties (example: natural catgut, hair ...).

In addition, the method according to the invention has another advantage in the context of its development. In fact, the techniques for producing microparticles are cumbersome and depend on many parameters in their development, in particular the surfactants used during their preparation. [28]. On the other hand, the method according to the invention uses a simple technique which consists in depositing antigens directly on a bioadherent, without the need for a sophisticated and expensive installation. Thus, the biological or chemical material is deposited in the presence of physiological liquid or after having undergone dehydration or lyophilization before being deposited on the surface of the bioadherent. Indeed, it is possible to dehydrate biological material while retaining its immunogenic character to make vaccines. THEURER K. [EP 0083 673 A1] uses, for preventive and therapeutic purposes, solutions, emulsions or dispersions based on dry powders (totally water-soluble and emulsifiable) from isolated organs or mixtures of organs. Many methods make it possible to strengthen the bonds between antigens and the support, within the framework of the method according to the invention, by increasing the number of chemical bonds. It is possible to spray antigens on the bioadherent or to impose pressure inside a pressurized enclosure, to enclose the antigens a little more on their support during the polymerization of the latter. This partial isolation allows, during the exposure time necessary to develop an immune response, to immobilize dangerous biological or chemical elements with a high degree of security, even when the material according to the process, is subjected to strong hemodynamic constraints or biomechanical. In the case of insertion into the tissues, the cyanoacrylates may be in the form of a film made porous. The porosity is obtained by using this bioadhesive in aerosol or by its degassing by means of a gas such as freon (fluorocarbon) during its polymerization. Porosity adds the added benefit of easier biodegradability, drainage and phagocytosis [29]. Therefore the cyanoacrylate polymer can be introduced in different porous forms according to the process of the invention.

Several techniques allow easy placement of the immunogenic material in the body at the level of a blood vessel or tissue.

<• - Insertion into a blood vessel. The most suitable form is the filament. This must be long enough for one part to remain inside the blood vessel while the other, outside the vessel, immobilizes the filament. The filament must be thin, regular, preferably flexible so as not to disturb the blood circulation, but also strong enough not to break during its stay in the blood vessel and during its removal. This antigen-coated filament is partially introduced into a blood vessel using a cannula or syringe (containing physiological saline, or a buffered solution, whether or not combined with an anticoagulant) and then withdrawn after use.

The time required for immunization by this method is relatively shorter than that required by standard immunization procedures such as injections. The most imperative constraint for an implant in contact with the blood is hemocompatibility, that is to say that it must not damage the proteins, the enzymes and the figured elements of the blood, and in particular not to create hemolysis and platelet reaction. The most important factor in hemocompatibility is coagulation, the mechanisms of which are still poorly understood and which depend on hemodynamic parameters ( blood shear forces on the surface of the material) imposing suitable geometries on the implant.

To remedy this aspect it is sometimes necessary to associate, by impregnation of the material, molecules (heparin, prostacyclin (PGI2)) or to graft on its surface chemical groups having an antithrombotic "heparin-like" function (sulfonamide groups or sulfonate).

Heparin remains the most powerful antithrombotic molecule. It improves the biocompatibility of many implants [30] and it can be combined with other molecules for complementary functions [31, 32]. The modification by heparinisation of the surface of polymers or copolymers intended for medical use has shown that in all cases it improves hemocompatibility [33, 34]. To optimize hemocompatibility, a general heparinization method applicable to all polymers has been developed and another more specific to polyurethanes although polyurethanes are a family of elastomers which have the best hemocompatibility properties compared to any other. type of polymer, [35].

Heparinization also relates to copolymers [36, 37]

There are processes using chemical groups expressing heparin (heparin-like) functions such as sulfonamides grafted onto polymers (polystyrene) or copolymers (polystyrenes / polyethylenes) [38]. Thus, to avoid any risk of thrombosis in the event of partial insertion of the material according to the method, into a blood vessel, several possibilities are possible such as:

- insert among the antigens, molecules or anticoagulant chemical groups,

- a specific form of the implant allowing the diffusion of an external source of anticoagulants connected to the external part.

<S> - Insertion into a fabric.

In comparison with insertion into a blood vessel, a wider range in the choice of its mode of insertion is allowed. The shape of the immunogenic bioadherent can vary depending on the mechanical properties of the tissue (lozenges, flexible or rigid filaments, needles, sticks, films ...) from the moment when the characteristics of the material create no trauma or tissue damage.

For example, the method according to the invention can be introduced into a blood vessel or into other tissues of the body by means other than the needle of a syringe or by a cannula. The material can be designed sufficiently rigid and provided with a pointed end acting as a penetration needle. The implant is then introduced either manually or by projection using a device provided for this purpose [39]. The method according to the invention can also be used in different variants which can be inserted into a fabric, for example:

- a patch lined with antigens according to the process and applied to a skin scarification,

- a patch bristling with one or more small needles treated according to the process and which can penetrate a tissue,

- a type of vaccinostyle comprising a slot for containing a film according to the process, - a needle or a type of vaccinostyle fitted with a shrink film according to the process which can be released into a tissue after removal.

The dimensions of the implant according to the method of the invention depend on the type of substrate chosen and on the size of the host which therefore remains to be determined empirically with conventional means. The method according to the invention has many advantages:

- It offers the advantage of presenting antigens on the surface of the bioadherent in different orientations. This may prove necessary in particular in the case of the fixation of certain cell types, most of the essential antigen sites of which are found both on the apical side and on the basal side associated with the extracellular matrix. A dispersion, on the surface of the bioadherent, of cells dissociated from a tissue by enzymatic treatment makes it possible to present to the immune cells a range of antigens hitherto inaccessible,

- It makes it possible to amplify the immune reaction by bringing together on the surface of the biomaterial one or more types of antigens to constitute different types of vaccines (mixed or polyvalent vaccines, stock-vaccine, autovaccine ...). Indeed the constitution of an amalgam of different antigens causes an amplification of the immune reaction. MARTIN- ONCINA F. [WO 97/02838] polymerizes between them antigenic proteins produced during a pathological process to induce a lymphocyte response of type TH2 (immunosuppressant), - 11 makes it possible to obtain an immune response for antigens of low molecular weight, which is difficult with conventional methods,

- It makes it possible to use reduced concentrations of antigens (from nanogram to picogram), which is far below the concentrations used in conventional methods. - It can also be applied to any other place in the body (dermis, endoderm, muscle ...) in the form of temporary or permanent implants.

- It avoids allergies often due to the toxic effects of certain adjuvants.

- It allows it to be used as a probe for analysis for diagnostic or research purposes after removal and study of the material implanted in the host organism. By way of example, the material can be coated with specific antibodies to detect in the blood the presence of particular biological organisms, of biochemical or chemical molecules. Authors have managed, in vitro, to separate a cell type in small quantities in a mixed solution containing other cell types in very high concentration, using polystyrene beads coated with antibodies [40].

- It makes it possible to be used as a biosensor for the “in vivo” analysis of biological or chemical interactions, if the antigens adhere directly (or via a conductive adherent) to a material itself conductive (polymers or conductive metals, optical fiber, etc.) connected to signal processing equipment, thus offering a new tool for biomedical research.

This immunization method according to the invention offers great potential in terms of vaccination, and well-founded hopes allow us to hope for rapid results in the fight against serious diseases other than infectious ones such as benign or malignant tumors or the development of metastases. Immunization by this method is carried out in a shorter time than conventional methods which require boosters to obtain the same level of antibodies. Furthermore, the repetition of vaccines to achieve perfect immunity for children or adults is not accessible materially or financially all over the world. Also, this invention makes it possible to avoid the drawback of repeated immunizations and their costs by optimizing the immune response by a limited exposure of the immunogens.

The method according to the invention is particularly intended for immunization and relates particularly to the biomedical field of the bio-technology or bio-engineering industries intended for human or animal health. BIBLIOGRAPHICAL REFERENCES

[I] - Langer R. and Folkman, J., Polymers for the sustained release of proteins and other macromolecules, 1976, Nature, 263, p. 797-800.

[2] - Saltzman W., Langer R., Transport rates of proteins in porous materials with known microgeometry, 1989, Biophysique Journal, 55, p. 163-171.

[3] - Cohen S. et al., Controlled delivery Systems for proteins based on poly (lactic / glycolic acid) microspheres, 1991, Pharm. Research 8, p. 713-720.

[4] - Ertl. HC. et al., Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines, 1996, Vaccine, 14, p. 879-885. [5] - Eldridge J., et al. Biodegradable and biocompatible poly (DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies, 1991, Immunology, 59, p. 2978-2986.

[6] - Lima SL, et al. Immunization by subcutaneous implants of polyester-polyurethane sponges coupled with antigen., Brazil Journal of Medical Biology Research 1999, 32, (4), p.443-447.

[7] - Davis S., Polymeric microspheres as drug carriers, Biomaterials, 1988, 9, p. 111-116.

[8] - Fauk W, et al., An immunocolloid method for the electron microscope,

Immunochemistry, 1971, 8, p.1081-1083.

[9] - Hainfeld J, Gold cluster-labeled antibodies, Nature, 1988, 333, p. 281-282. [10] - Shibuya T.Y. et al., Anti-CD3 / anti-CD28 bead stimulation overcomes CD3 unresponsiveness in patients with head and neck squamous cell carcinoma., Arch

Otolaryngology Head Neck Surgery, 2000, 126 (4), p. 473-479.

[II] - Olivier JC, et al., Indirect evidence that drug brain targeting using polysorbate 80- coated polybutylcyanoacrylate nanoparticles is related to toxicity. Pharmaceutical Research, 1999, 16 (12), 1836-1842.

[12] - Hamada J, et al., Embolization with cellulose porous beads, TJ: Clinical trial. AJNR

American Journal of euroradiology, 1996, 17 (10), 1901-1906.

[13] - Retzinger GS, Dissemination of beads coated with trehalose 6,6'-dimycolate: a possible role for coagulation in the dissemination process Experimental Molecular Pathology, 1987, 46 (2), 190-198.

[14] - Yokel RA et al, Acute toxicity of latex microspheres, Toxicology Letter, 1981, 9 (2),

165-170.

[15] - Noordzij JP et al., Tissue adhesive wound repair revisited, Journal of Emergency

Medicine, 1994, 12 (5), p. 645-649. [16] - Quillen DA et al., Aerosol application of cyanoacrylate adhesive, J Refract Corneal

Surg 1994, 10 (2) p. 149-150.

[17] - Cuschieri A, Tissue adhesives in endosurgery, Semin Laparosc Surg 2001, 8 (1), p.

63-68. [18] - King ME et al., Tissue adhesives: a new method of wound repair Nurse Pract 1999,

24 (10), p. 66, 69-70, 73-74.

[19] - Vargas G et al., An alternative to sutures, Medsurg Nurs., 2000, 9 (2), p.83-85.

[20] - De Paulis R et al., Cyanoacrylate glue as an alternative to an additional suture line in the repair of type A aortic dissection, Texas Heart Institute Journal, 1999, 26 (4), p. 275-277. [21] - Marcovich R et al. , Comparison of 2-octyl cyanoacrylate adhesive, fibrin glue, and suturing for wound closure in the porcine urinary tract. Urology 2001; 57 (4), p. 806-810.

[22] - Schroeder U et al, Diffusion enhancement of drugs by loaded nanoparticles in vitro,

Prog Neuropsychopharmacol Biol Psychiatry, 1999, 23 (5), p. 941-949.

[23] - Sai P, et al., Prophylactic oral administration of metabolically active insulin entrapped in isobutylcyanoacrylate nanocapsules reduces the incidence of diabetes in nonobese diabetic mice, Journal of Autoimmunity, 1996, 9 (6), p. 713-722.

[24] - O'Hagan DT, Palin KJ, Davis SS.Poly (butyl-2-cyanoacrylate) particles as adjuvants for oral immunization, Vaccine 1989, 7 (3), p. 213-216.

[25] - Simeonova M et al. Study of the effect of polybutyl cyanoacrylate nanoparticles and their metabolites on the primary immune response in mice to sheep red blood cells.,

Biomaterials, 1998,19 (23), p. 2187-2193.

[26] - Chattaraj SC et al., Biodegradable microparticles of viral influenza vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice, Journal of Control Release, 1999, 29; 58 (2), p. 223-232. [27] - Illum L et al., Tissue distribution of poly (hexyl 2-cyanoacrylate) nanoparticles coated with monoclonal antibodies in mice bearing human tumor xenografts., Journal of

Pharmacol Exp Ther, 1984, 230 (3), p. 733-736.

[28] - Nandelli MA et al., An interpretative analysis of the effect of the surfactants used for the preparation of polyalkylcyanoacrylate nanoparticles on the release process, Journal of Microencapsulation, 1994, Oct; ll (5), p. 531-538.

[29] - Anony., COAPT tissue adhesive aerosol, Investigational New Drug Information

Brochure, Ethicon, Inc, Somerville, N.J., Jan 1969.

[30] - Tatarkiewicz K. et al, In vitro and in vivo eveluation of protamine-heparin membrane for microencapsulation of rat Langerhans islets, artificial Organs, 1994, 18 (10), p. 736-739. [31] - Bryanda E, et al., Albumin and heparin multylayer coatings for blood-contacting medical devices, Journal of Biomedical Research, 2000, 51 (2), p. 249-257.

[32] - Goodwin S. et al, Percutaneous delivery of a heparin-impregnated collagen stent-graft in a porcine model of atherosclerosis desease, Investigation Radiology, 2000, 35 (7), p. 420-425.

[33] - Guess C „Heparin-bonded Dacron or polytetrafluoroethylene for femoropopliteal bypass grafting: a multicenter trial, Journal of Nascular Surgery, 2001, 33 (3), p. 539.

[34] - Shin EK et al., Efficacy of heparine-coated stent in early setting of acute myocardial infarction, Cardiovascular Intervention Catheter, 2001, 52 (3), p. 313. [35] - Ito Y., Antithrombognic heparin-bound polyurethane, Journal of Biomaterials

Application, 1987, 2 (2), p. 235-265.

[36] - Miyama H. et al., Antithrombogenic heparinized polyacrylonitrile copolymer, Journal of Biomedical Material Research, 1986, 20 (7), p. 895-901.

[37] - Tanzi et al., Heparinizable graft copolymers from chlorosulphonated polyethylene with poly (amino-anine) segments, Biomaterials, 1985, 6 (4), p. 273-276.

[38] - Lautier A., ES A studies on heparin-like materials used for extracorporeal shunts,

International Journal of Artificial Organs, 1982, 5 (3), p. 199-204.

[39] - Kesler DJ, et al., Efficacity of sustained release needle-less ceftiofur sodium implants in treating calves with bovine repiratory disease, Journal of veterinary Medicine, B, 1999, 46, p.25-35.

[40] - Gomez SM et al. Capture of rare cells in suspension with antibody-coated polystyrene beads.Biotechnology Progress 1999, 15 (2), p. 238-244.

The accompanying drawings illustrate the invention:

Particular embodiments of the invention will be described below, by way of purely indicative and in no way limitative examples.

Figure 1.

- Figure la shows a section of an immunogenic implant according to the invention the invention (1) consisting of a bioadherent (2) on the surface of which antigens (3) are fixed irreversibly.

- Figure lb shows a section of an immunogenic implant according to the invention (1) consisting of a bioadherent (2)) on the surface of which antigens the cells of a tissue (4) are fixed irreversibly. The bioadhesive is spread over a material (5).

Figure 2.

- Figure 2 shows a means of insertion into the body of the immunogenic implant (1) according to the method. The implant is introduced via a pointed end (6) acting as a penetration needle.

Figure 3.

- Figure 3 shows a variant of the process in the form of a patch (7) with immunogenic tips according to the process (8) inserted into the skin (9).

Claims

1) The present invention relates to a new immunization method consisting in inducing or stimulating an immune reaction in humans or animals by inserting an immunogenic implant consisting of a chemical adherent for biological use, or bioadherent, on the surface. from which antigens of biological origin (tissue, cells, microsomes, microorganisms, natural or synthetic biological molecules) or of chemical origin (simple molecules or in the form of complexes, chemical functional groups) are fixed irreversibly. The immunogenic implant thus formed is inserted either into a blood vessel, partially and temporarily, or into a tissue, partially or completely, temporarily or permanently. 2) Method according to claim 1 characterized in that the bioadhesive is designed from polymers, polymer resin or silicone. 3) Method according to claim 1 and 2 characterized in that the bioadhesive establishes irreversible covalent bonds with the antigens to be bound, during its polymerization. 4) Method according to claim 2 and 3 characterized in that the bioadhesive is a polymer designed from cyanoacrylates (akyl-α-cyanoacrylate, methyl- and ethyl-2-cyanocrylate, cyanoacrylate 2-n-butyl) porous or not. 5) Process according to claim 1 to 4 characterized in that the bioadhesive is previously spread over a material of variable nature: carbon, organic compounds comprising: synthetic polymers or copolymers made from different families of polymers (polyolefins, vinyl, styrenics, acrylics, polyamides, saturated polyesters, polycarbonates, polyacetals, fluoropolymers, silicones, polyurethanes), silicones, natural biopolymers (cellulose, silk ...), modified natural biopolymers (e.g. polysaccharide with a carboxylic function. ..), artificial biopolymers (ex: homopolypeptide ...) or artificial biocopolymer (ex: lactic acid and gly colic copolymer), non-organic materials including the appropriate metals (gold, silver, nickel, platinum ...) ) and alloys, ceramics, fabrics treated specifically to give them the essential biomechanical properties (example catgut natural, hair ...). 6) Method according to claim 1 characterized in that the biological or chemical material is deposited in the presence of physiological liquid or after having undergone dehydration or lyophilization before being deposited on the surface of the bioadherent. 7) A method according to claims 1 and 5 characterized in that the bonds between antigens and the support are reinforced by increasing the number of chemical bonds in imposing a pressure inside a pressurized enclosure so as to enclose the antigens a little more on their support during the polymerization. 8) Method according to claim 1 characterized in that the implant (antigen fixed on bioadherent) has a shape can vary depending on the mechanical properties of the tissue (size and geometry) and not allowing its diffusion in different tissues (pellets, needles, flexible or rigid filaments, rods, films ...). 9) Method according to claim 1 and 8 characterized in that the immunogenic implant is film fitting a needle or a vaccinostyle can be dropped in a tissue. 10) Method according to claim lcaractérisé in that the immunogenic implant is contained in the slot of a vaccinostyle 11) A method according to claim 1 characterized in that molecules or anticoagulant chemical groups are inserted among the antigens when the immunogenic implant is temporarily inserted into a blood vessel. 12) Method according to claim 1 characterized in that an external source of anticoagulants is connected to the external part of the immunogenic implant is temporarily inserted into a blood vessel. 13) Method according to claim 1 characterized in that the immunogenic implant is applied in the form of a patch on a skin scarification 14) Method according to claim 1 characterized in that the immunogenic implant is applied in the form of a patch bristling with one or more small needles which can penetrate into a tissue