MXPA99010730A - Immunopotentiating composition - Google Patents

Abstract

The present invention provides an immunopotentiating composition which comprises an antigen or antigen-inducing substance, and a carrier comprising a biocompatible material for effectively increasing an immune response derived from an antigen. The present invention further provides a method of producing an antibody by administering said immunopotentiating composition to a mammal or bird, thereby modulating the immune response in said mammal or bird and recovering the antibody produced.

Description

IMMUNOPOTENTIATING COMPOSITION TECHNICAL FIELD The present invention relates to an immunopotentiating composition for effectively increasing an immune response derived from an antigen. The immunopotentiating composition according to the present invention is used primarily as a vaccine preparation in the field of human medicine or veterinary medicine for the purpose of preventing or treating diseases in humans, in mammals other than humans, and in birds. In addition, the immunopotentiating composition according to the present invention is used to immunize animals for the purpose of producing antibodies.

TECHNICAL BACKGROUND The vaccines that are currently in general use are classified into two groups, attenuated vaccines (live) and inactivated (destroyed) vaccines. Attenuated vaccines are advantageous because good immune responses can generally be obtained, but they are disadvantageous because, from a safety point of view, there are anxiety factors such as restoration of toxicity and adverse effects therewith. The inactivated vaccines are safer compared to the attenuated vaccines, but they are disadvantageous because the individual administration thereof can hardly produce a sufficient immunizing effect. In fact, in preventive vaccination with inactivated vaccines, two or more administrations are carried out at intervals of 2 to 3 weeks, in order to obtain a satisfactory effect. Meanwhile, recent advances in the methodology of molecular biology have made it possible to identify disease-specific antigens useful in the prevention or treatment of diseases, and to produce synthetic antigens (component vaccines), simulating antigens identified by the use of chemical technology or of recombinant DNA. The antigens synthesized in this way are superior in purity, stability, specific character and safety, to conventional vaccine antigens. However, from the practical point of view, they are of generally low antigenicity, and this is the biggest problem that will be solved. The arrival of a method to efficiently produce an antibody for an antigen having low antigenicity is thus seriously desired from the standpoint of human and veterinary medicine. Furthermore, when an inactivated vaccine or component vaccine is used, it is necessary, to achieve an antibody production degree that is effective for the prevention or treatment of a disease, to make 2 or 3 administrations at intervals of 2 to 3 weeks, preferably 4 weeks or more. Therefore, a vaccine capable of producing a sufficient effect after an individual administration (individual dose vaccine), is seriously demanded in human and veterinary medicine. In the field of veterinary medicine, its main advantages include 1) time reduction, 2) cost reduction and 3) improved compliance. In human medicine, too, the three advantages mentioned above are important and, among them, improved compliance is particularly important in developing countries, where a plurality of administrations with observed intervals is virtually impossible. The problem of the weak antigenicity of inactivated vaccines and component vaccines can be solved, at an experimental level, by the use of an adjuvant. However, in practice, this solution has several problems such as adverse effects. The technology of adjuvants that use artificial substances includes two methods. One comprises dispersing an antigen on the surface of oil or lipid particles, and the other comprising causing an antigen to be adsorbed onto a precipitate. For some time, mineral oil had been used for veterinary vaccines or military influenza vaccines, causing severe hemorrhagic lesions or extensive granuloma. Since then, their common use in vaccines for human use has never been approved by the relevant authorities. Freund's complete and incomplete adjuvants were used over the last 4 decades more extensively in animal experiments. Neverthelessalthough they induce the production of antibodies to a satisfactory degree, they promote the adhesion and formation of granulomas at the site of infusion, pyrexia and other toxic effects and, consequently, the use thereof has been avoided in human or veterinary medicine. Alum, aluminum hydroxide or aluminum phosphate, is currently the only adjuvant approved for administration in humans and is widely used. However, it causes granuloma formation at the vaccination site and, in addition, its effect varies widely in a case-by-case disadvantageous manner. For example, aluminum hydroxide produces a sufficient adjuvant effect with bacterial toxoids but, with vaccines against hepatitis B virus or influenza virus, good results have not been obtained. In addition to the adjuvant techniques mentioned above that use artificial substances, there is a method that uses, as an adjuvant, a cytokine that originally occurs in the living body and that has immunoactivation activity. In fact, it is reported or described that the production of antibodies in response to antigens is increased by the use of cytokines that have immunoactivation activity (eg, IL-1, IL-2, IFN- ?, IFN-, GM- CSF, IL-12, etc.). These findings are reviewed, for example, by Rong Lind et al. (Clinical Infection Diseases, 21, 1439-1449, 1995). However, when a cytokine is used as an adjuvant in the dissolved state, a plurality of administrations are required to achieve a satisfactory effect of antibody production, although its adverse effects are scarce and less severe compared to the artificial adjuvants mentioned above. It is assumed that as an organism is inoculated with an antigen and a cytokine in the dissolved state, they are diffused in the body immediately after inoculation, thus not being able to activate the specific immunomechanism for the antigen. Furthermore, for systemic immunoactivation with a cytokine, the cytokine is required in large quantities and, in that case, several adverse effects may possibly be induced. Therefore, a method is required to effectively use a cytokine as an adjuvant without causing adverse effects. As an additional alternative to the solution to the problem of low antigenicity of inactivated vaccines and component vaccines, the technique of sustained release of the vehicle's antigen can be mentioned. The idea of sustained release of the antigen arises from the view that the effect of the adjuvant obtained with alum is due to the non-specific adsorption of the antigen on the alum, and the sustained release thereof from the latter. Hitherto, attempts have been made using various vehicles (for example, Bongkee Sah et al., J. Pharm. Pharmacol., 48, 32-36, 1996), but none has had practical use. The period of administration of the antigen to the production of antibodies is also very important from the point of view of prevention or treatment of the disease. However, until now there have been no attempts to shorten this period required for the production of antibodies.

In view of the foregoing, the following are preferred objects of the present invention: (1) To provide an immunopotentiating composition with which an antigen is, or an antigen and a substance having immunoactivation, immunostimulation or immunomodulation activity is, released ( s) sustainably of a vehicle comprising a biocompatible material; (2) To provide an immunopotentiating composition with which an antigen-producing substance is, or an antigen-producing substance and a substance having immunoactivation, immunostimulation or immunomodulation activity is, sustained release from a vehicle comprising a biocompatible material; (3) Provide a method to increase the immune response derived from an antigen, using the provided composition achieving the objectives (1) or (2) above, without causing adverse effects; (4) Provide a method for reducing the period required for the production of antibodies derived from an antigen, using the composition provided by achieving objectives (1) or (2) above; (5) Provide a method for prolonging the period of immunity derived from an antigen, using the composition provided by achieving objectives (1) or (2) above; (6) Provide a method for achieving immunopotentiation using the provided composition by achieving objectives (1) or (2) above, the surroundings of said composition being the sites of immune response; (7) Provide a vaccine for human use and for use in mammals other than humans and birds, using the composition provided by achieving objectives (1) or (2) above; (8) Provide an individual dose vaccine for use in humans and for use in mammals other than humans and birds, using the composition provided by achieving objectives (1) or (2) above.

Means for solving the problems The present inventors made intensive investigations in an attempt to obtain a composition that allows the sustained release of antigens from a biocompatible material and, as a result, unexpectedly found that when an immunopotentiating composition comprising a biocompatible material and an antigen carried on it, it is administered to living organisms, the immune response derived from the antigen is increased. In addition, the present inventors found that the administration, to the living body, of an immunopotentiating composition comprising a substance having immunoactivation, immunostimulation or immunomodulation activity (collectively referred to collectively herein as "immunomodulatory substance") carried simultaneously on a carrier comprising a biocompatible material together with an antigen, results in the production of an early and also improved immune response. Based on these findings, the present invention has now been concluded. Next, the present invention is described in greater detail. i) Explanation of the principle of the present invention In typical humoral immune responses, the production of antibodies subsequent to the second stimulation with an antigen occurs earlier and at a higher antibody titer, which is maintained for a longer period, compared with the production of antibodies consecutive to the first stimulation with the antigen. The difference in the period required for the production of antibodies is due almost to the fact that while the first antigenic stimulation will be followed by a series of steps, namely (1) presentation of the antigen to T cells by cells presenting antigen and activation of T cells, (2) activation of B cells by activated T cells, (3) transport of antigen to lymph nodes by dendritic cells and (4) proliferation of B cells in lymph nodes and differentiation of these into cells that form antibodies , a sufficient number of antibody-forming cells are already available at the time of the second stimulation. The difference in the level of antibody titer and in the duration of high antibody titers is due to the fact that, in immunological stimulation using the conventional solution form, the antigen administered for the first immunological stimulation is diffused throughout the body. , and it is degraded, metabolized and eliminated. The antigen has disappeared mainly from the body when the cells that form antibodies are prepared for the production of the same; therefore, there is no stimulation again of the cells that form antibodies by the antigen. To achieve a higher antibody titer or longer duration, which is important for the prevention or treatment of a disease, it is therefore necessary that the cells that form antibodies produced in response to the first antigenic stimulation, be stimulated again by the antigen. On the other hand, the earlier production of antibodies is also important in the prevention or treatment of diseases. For the earlier production of antibodies, it is important to induce the efficient production of antibody-forming cells by the first antigenic stimulation. For such efficient production of antibody-forming cells, it is necessary to (1) increase the chances of contacting the antigen with antigen-presenting cells (by causing antigen-presenting cells to accumulate at the site of administration thereof), and (2) ) increase the activation of B cells and the differentiation of these into cells that form antibodies in lymph nodes. With these points in mind, the present invention obtained higher antibody titers of greater duration by causing an antigen to be stably carried on a vehicle comprising a biocompatible material and being released therefrom in a sustained manner to maintain the amount of antigen in the body, thereby that the antigen stimulates again the produced cells that form antibodies. In particular, although it can be easily estimated that the concentration of antigen is maintained at a high level in and around the site of administration of the immunopotentiating composition, this state of high and local antigen concentration promotes the reaction between the antigen and the cells that they form antibodies, which is an equilibrium reaction and, at the same time, cause the accumulation of immunocompetent cells in and around the site of administration. Therefore, maintaining the local concentration of antigen at a high level can be mentioned as the most important principle of the present invention. In addition, the present invention achieved earlier and more efficient antibody production by causing an antigen and an immunomodulatory substance (eg, cytokine) to be simultaneously carried on a vehicle comprising a biocompatible material, and to be released therefrom in a sustained manner so as to (1) promote the local accumulation of immunocompetent cells in and around the site of antigen administration and the subsequent activation of antigen presentation to B cells, and (2) increase the activation of B cells and their differentiation into cells that form antibodies in the lymph node, in charge of the site of administration of the immunopotentiating composition (the lymphatic node to which the dendritic cells transfer the antigen and in which cells that form antibodies are produced) through the selective and continuous entry of the cytokine in said 1 lymphatic node Therefore, a characteristic feature of the immunopotentiating composition according to the present invention is that, unlike the mechanism of systemic immunoactivation induced by the administration of an antigen, or an antigen and a cytokine, in the form of a solution, an immunopotentiation field is formed around the composition through the sustained release of the antigen, or the antigen and the cytokine. In view of the foregoing, the preferred features of the present invention can be summarized as follows: (1) The antigen or antigen-inducing substance (collectively referred to herein as "antigenic substance") and, when present, the The immunomodulatory substance can be released sustainably. (2) The concentration of the antigenic substance and, when present, the immunomodulatory substance, can be maintained at a high level at the site of administration. These characteristics can be provided by stably maintaining, in the living organism, the antigenic substance or the antigenic substance and the immunomodulatory substance, which are carried or sustained on a vehicle constituting the immunopotentiating composition, and allowing said substances to be released sustained in said organism. Since the immunopotentiating composition is administered to living organisms, it is indeed required that the vehicle be a material having good biocompatibility. Thus, when a biocompatible material capable of satisfying the above requirements imposed from the pharmaceutical point of view is used as a carrier, an immunopotentiating or immuno-enhancing effect can be produced using the resulting immunopotentiating composition, regardless of which biocompatible material is used, and without contradict the principle of the present invention. In addition, these fundamental features of the present invention can be mentioned not only with respect to humoral immunity, but also with respect to mucosal immunity and cell-mediated immunity since, in each case, immunity is activated as a result of stimulation. Continuous antigenicity of immunocompetent cells and the positive acceleration of T and B cell activation. ii) Efficates of the present invention The effects produced by the present invention are mentioned below. a) Sustained release of an antigen or of an antigen and a cytokine As shown in Figure 1, an immunopotentiating composition of the present invention delivered an antigen (avidin) and a cytokine (IL-1β) sustained for 7 days or more . b) Increase in antibody production The increasing effect of the production of antibodies of the immunopotentiating composition of the present invention was established in immunological stimulation experiments in mice and sheep. In this manner, avidin was administered in a varied dosage form to mice, and antiavidin antibody titers in blood were determined by the ELISA technique at 7, 14, 21, 35 and 83 days after administration (Figure 2). ). They were compared with some other case, the case in which 100 micrograms of avidin were administered to mice in a conventional manner, namely, in the form of a solution of avidin in phosphate pH regulator, the case in which a composition was administered immunopotentiator (prepared in example 7) having the same amount of avidin, and the case in which an immunopotentiating composition (prepared in example 8) having the same amount of avidin was administered simultaneously with IL-1β. At 35 days after the administration, the antibody titer in the blood of the mice to which the avidin-possessing immunopotentiating composition was administered was approximately 25 times higher, compared to the antibody titer obtained in the mice to which it was added. I administered the avidin solution. This result indicates that the use of the antigen in the immunopotentiating composition in accordance with the present invention, resulted in the increased production of antibodies in response to the antigen. In addition, in mice given the immunopotentiating composition possessing avidin and IL-1β, the antibody titer was as high as approximately 450 times the antibody titer obtained after administration of the avidin solution. This result indicates that the production of antibodies in response to an antigen can be further increased by the use of the immunopotentiating composition of the present invention which simultaneously contains an antigen and a cytokine and which has immunoactivation activity. The effect of the production of antibodies of the immunopotentiating composition, which simultaneously possesses an antigen and an immunoactivation cytokine, was observed more markedly in an immunostimulation experiment in sheep (Figure 3). Avidin was administered in various dosage forms to sheep, and antiavidin antibody titers in blood were determined by the ELISA technique at 7, 14, 21 and 35 days after administration. When avidin was administered as a conventional solution, the production of antiavidin antibodies was not confirmed even at a dose of 100 micrograms. This difference in the production of antibodies between mice and sheep is supposedly due to the difference in body weight. This indicates that 100 micrograms of avidin do not have sufficient antigenicity to cause the production of antibodies in sheep. In contrast, when an immunopotentiating composition (prepared in Example 8) possessing avidin and IL-1β was simultaneously administered, a high level of antibody production was established at 14 days after administration.

This result clearly indicates that the immunopotentiating composition of the present invention has an increasing effect on the production of antibodies in response to an antigen that has hardly sufficient antigenicity to cause the production of antibodies. In contrast, when avidin and IL-1β were administered simultaneously in the conventional solution form, no antibody production was detected. This result indicates that the enhancing effect of antibody production produced by the immunopotentiating composition depends on the sustained release of the antigen and the cytokine. The above finding indicates that in contrast to the conventional method of administration which requires a plurality of administrations to achieve a sufficient antibody titer, the immunopotentiating composition of the present invention can give a sufficient antibody titer only after administration. c) Reduction of time to the production of antibodies The reduction of time to the production of antibodies, which is one of the important effects of the immunopotentiating composition of the present invention, was directly investigated in the immunostimulation experiment using mice, in the which even after the administration of the antigen solution in conventional manner, a certain degree of antibody production was observed, rather than in the immunostimulation experiment using sheep in which the administration of the antigen solution in a conventional manner could not induce the production of antibodies. As is clear from the graphic representation of Figure 2, when avidin was administered as a solution in conventional manner, the anti-avidin antibody titer increased from day 14 after administration, whereas when the composition was administered immunopotentiator (carrying avidin alone or avidin plus IL-1β), the amount of antibodies showed a rapid increase from the seventh day after administration, whereby the time to antibody production was reduced by about a week. 83 days elapsed after the administration, so that the antibody titer in mice given avidin as a solution would reach the same antibody titer obtained on day 14 after administration of the immunopotentiating composition possessing avidin alone . With the avidin solution, the antibody titer obtained on day 14 after administration of the immunopotentiating composition possessing avidin and IL-1β could not be achieved even on day 83 after administration. In this regard, it can be said that the immunopotentiating composition shortened the period required for the production of antibodies by at least 69 days. d) Immune response in the vicinity of the administration site of the immunopotentiating composition As already mentioned in the section "explanation of the principle of the present invention", the immune response that occurs locally in the vicinity of the administration site seems to play an important role in the effect obtainable with the immunopotentiating composition of the present invention. This can be easily investigated from the histological panorama at the administration site of the sheep used in the immunostimulation experiment (Figures 4, 5). Tissue microfog- raphy shows the infiltration of immunocompetent cells around the immunopotentiating composition. The immunocompetent cells as referred to herein, include neutrophils, CD4 positive T cells, T cells. and d TCR positive, MHC II positive cells, macrophages, etc. Said accumulation of immunocompetent cells was not observed when avidin and / or IL-1β were administered in the form of a solution. This is possibly due to the immediate diffusion in the body, after inoculation, of avidin and IL-1β in solution form, resulting in the impossibility of guiding the immunocompetent cells to the site of inoculation. The accumulation of immunocompetent cells was more conspicuous with the immunopotentiating composition possessed by IL-1β. These findings support the view that a state of high concentration of antigen, or antigen and cytokine, is created around the immunopotentiating composition as a result of the sustained release of said composition, thus resulting in increased antibody production through of the accumulation of immunocompetent cells around said composition.

On the other hand, the accumulation of immunocompetent cells is a type of inflammatory response. However, the inflammatory response evoked was not accompanied by edema or its similar, and stopped by itself. iii) The immunopotentiating composition The term "immunopotentiating composition", as used herein, means, in principle, a composition or preparation comprising a vehicle, which is a biocompatible material, and an antigen or antigen-inducing substance carried on said vehicle and, if desired, further comprising an immunomodulatory substance, as described below, and / or one or more pharmaceutical additives. The "immune response" that will be enhanced by the immunopotentiating composition of the present invention, is the specific immune response to the antigen contained in said composition, or the antigen induced by the inducer contained therein. The immune response that will be activated may be humoral immunity, mucosal immunity or cellular immunity, or a combination thereof. The term "antigen" is not limited to any particular species, as long as it can induce the antigen-antibody reaction derived from the antigen. In general, it is selected from those antigens to which antibodies are produced effective in the prevention and / or treatment of diseases in humans or mammals other than human, or in birds. This way includes, but is not limited to, those toxoids, vaccines and vaccines. live vaccines themselves that are described, for example, in "Vaccine Handbook" (edited by the National Institute of Health Alumni Association, published by Maruzen Co.), "Immunizing Agents", in Remington's Pharmaceutical Sciences, fourteenth edition, 1990, Mack Publishing Co., section 75, pages 1426-1441, or Physician's Desk Reference to drugs, approved by the United States Food and Drug Administration, edition 46, pages 208-209, 1992. In addition, it includes, but is not limited to, following: (1) Virus, mycoplasma, bacteria, parasites, toxins, tumor cells and the like attenuated or made non-toxic or non-pathogenic, for example by gene recombination (modification of the gene related to toxicity or pathogenicity), continuous subculture (appearance of attenuated or nonpathogenic strains as a result of self-modification), treatment with formalin, treatment with β-propiolactone, exposure to radiation, sound treatment, treatment with enzymes, or similar. (2) Proteins such as membrane surface proteins and nuclear proteins, proteoglycans, polypeptides, peptides, membrane components and the like Qbtenids of viruses, mycoplasmas, bacteria, parasites, toxins, tumor cells and the like, for example, by chemical degradation or enzymatic, physical dissolution, column purification, extraction or filtration. (3) Vaccines of subunits, synthetic peptides having a sequence such that they are comparable or superior in specific antigenicity to the corresponding antigens, and the like, as obtained by cutting off a gene encoding an antigen capable of inducing virus-specific immunity, mycoplasma, bacterium, parasite, toxins, line of tumor cells or the like from the virus, microplasma, bacterium, parasite, line of tumor cells or the like, identifying said gene, inserting it in an appropriate vector such as a plasmid, and causing the gene is expressed in Escherichia coli, yeast or animal cells. The antigen capable of inducing a specific immune response to tumor cells, as referred to herein includes, but is not limited to, so-called tumor regression antigens such as MAGE-1, MAGE-3 and BAGE, tissue-specific antigens such as tyrosinase, Mart-1, gp100 and gp75 and, in addition, p15, Mucl, CEA, HPV E6, E7, HPR2 / neu, and the like. The "antigen" includes, but is not limited to, antigens capable of inducing an immune response responsible for the onset of, or effective in the treatment of, diseases as mentioned below: cholera, whooping cough, plague, typhoid fever, meningitis, pneumonia, leprosy, gonorrhea, dysentery, polio, gram-negative sepsis, colibacilemia, rabies, diphtheria, botulism, tetanus, poliomyelitis, influenza, Japanese encephalitis, rubella, measles, yellow fever, mumps, hepatitis A, hepatitis B, hepatitis C, chicken pox, herpes zoster, malaria, tuberculosis, candidiasis, dental caries, acquired immunodeficiency syndrome, cancer (tumors) , matitis, anthrax, brucellosis, lymphadenitis caseosa, enterotoxemia, enteritis, black disease, malignant edema, black leg, leptospirosis, scabby mouth, vibriosis, erysipelas, gurma, Bordetella bronchitis, distemper, panleukopenia, rhinotracheitis, viral diarrhea and Pimelea poisoning . The antigen also includes, but is not limited to, antigens capable of inducing an effective immune response in the prevention of infection with said virus, mycoplasma, bacteria or parasite as mentioned below, in preventing the onset of the relevant disease, and in the treatment of patients with said disease: Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, salmonella, group B meningococcus, group B streptococcus, adenovirus, coronavirus, RS virus, human immunodeficiency virus I and II, herpes simplex I and II , CMV, EBV, Chlamydia trachomatis, parvovirus, parainfluenza virus, calcivirus, etc. The "antigen" also includes antigens which are not only used for animal health, but also for animal reproduction. For example, the antigen is described in "Vaccines in Agriculture, Immunological Applications to Animal Health and Production" (edited by P. R. Wood et al., CSIRO, 71-160, 1994). The antigen includes, but is not limited to, antigens used for animal reproduction as mentioned below: 1) antigens for reproduction; antigens that can induce immunoresponse against peptides related to inhibin and releasing hormones, such as luteinising hormone releasing hormone, gonadotrophin releasing hormone, etc .; 2) antigens for the control of the growth and metabolism of animals; antigens that can induce immune response against factor related to growth hormone, insulin-like growth factor-1, growth hormone, steroid hormones, sex steroid hormones, adipocyte plasma membrane antigens, lipids, cortisol, adenocorticotrophic hormone, hormone receptor adenocorticotrophic, ß-adrenergic receptor, adenohypophyseal hormones such as prolactin, ACTH, STH, TSH, LH, FSH, etc .; 3) antigens for environmental control; and 4) antigens that can induce immune response against plant-associated toxins, and low-molecular-weight natural toxicants. In addition, the term "antigen" is not limited to any particular species, as long as it can induce a specific immune response to antigens, and also includes antigens capable of inducing a non-specific immune response to antigens. The antigen capable of inducing non-specific immune response to antigens, as referred to herein, includes, but is not limited to, so-called superantigens such as staphylococcal enterotoxins, toxic shock syndrome toxin 1, exofoliative dermatitis toxin, CAP ( cell membrane associated protein) and SPM (Streptococcus pyogenes mitogen), such as T-12 and NY-5, described in Miyagiken Ishikai Kaiho, Vol. 50, 133-137, 1997, and the like. The term "antigen-producing substance" means a substance capable of inducing said antigen in vivo as mentioned above and includes, inter alia, plasmids and viruses containing a nucleic acid encoding a gene sequence for an antigen capable of inducing specific immunity to a virus, mycoplasma, bacteria, parasite, toxins, tumor cells or the like as inserted herein, so that the relevant antigen can be produced in vivo. The nucleic acid to be inserted includes, but is not limited to, nucleic acids encoding substances capable of functioning as antigens as mentioned above, for example, nucleic acids encoding the following proteins: HA or NA protein or NP Influenza, E2 or NS1 protein of hepatitis C virus, hepatitis B virus type HBs antigen protein, VP1 or VP3 capsid protein of hepatitis A virus, capsid proteins, Egp virus protein dengue, the F or G protein of the RS virus, the structural protein G or N of the rabies virus, the gD protein of the herpes virus, the E1 protein or pre-M of the Japanese encephalitis virus, the cover protein VP4 or VP7 cover protein of rotavirus, gp120 or gp160 protein of human immunodeficiency virus, Leishmania main surface antigen protein, antigen core surface antigen protein orozoite of the malaria cycle, the 54-kd protein or CS of Toxoplasma, the cell-surface PAc protein of Streptococcus mutants that cause caries, such as tumor regression antigens such as MAGE-1, MAGE-3 and BAGE, tissue-specific antigens such as tyrosinase, Mart-1, gp100 and gp75, nucleic acids encoding p15, Muc1, CEA, HPV, E6, E7, HPR2 / neu, etc., and those nucleic acids described in "Immunization with DNA "; Journal of Immunological Methods, vol. 176, 1994, pages 145 to 152.

Plasmids or viruses in which said nucleic acid will be inserted are not limited to any particular species, as long as they are non-pathogenic. Thus, like viruses, there may be mentioned those viruses which are generally used as vectors in gene therapy, for example adenoviruses, adeno-associated viruses, vaccinia viruses, retroviruses, HIV viruses and herpes viruses. The antigenic substance can be obtained using chemical, recombinant DNA, fermentation or cell culture technology. In the practice of the present invention, the method for preparing said substance is not limited to any specific method. However, since the composition of the present invention has the effects mentioned above, said antigens which are particularly suitable for use and which are obtained by recombinant DNA technology, are thus of low antigenicity and, in general, can be produced hardly effective after its administration by the conventional method (for example, parenteral administration in the state of solution or suspension). The antigenic substance for inducing specific immunity can be incorporated as such in the immunopotentiating composition, without any modification or, to further increase its antigenicity and / or increase its stability it can be, for example, (1) covalently or non-covalently linked to a protein having a molecular weight greater than the antigen, for example β-galactosidase or a nuclear protein, (2) supplemented with an appropriate chain of sugars (carbohydrates), (3) included in liposomes, (4) included in liposomes of type virus fusion-liposome membrane, or (5) contained in virosomes obtained by the use of B30MDP [6-O- (2-tetradecyl hexadecanoyl) -N-acetylmuramyl-L-alanyl-D-isogyutamine]. The "immunomodulatory substance (substance having immunoactivation, immunostimulation or immunomodulation activity)" is not limited to any particular species but includes, among others, cytokines, chemokines, growth factors, DNA sequences and adjuvant peptides, alum, Freund's complete adjuvant, incomplete Freund's adjuvant, iscom, saponins, hexadecylamine, dimethyldioctadecylammonium bromide, Abridin, skeletal components of the cell wall, cholera toxin, lipopolysaccharide endotoxins, liposomes that include liposomes containing cytokine, and liposomes by Walter Reed , 1, 25-dihydroxyvitamin D3, and gelation products from a polymer of carboxylvinyl, alginin and sodium chloride. The "cytokine" is not limited to any particular species, as long as it has immunoactivation activity, including in this manner, among others, IFN-a, IFN-β, IFN- ?, IL-1 a, IL-1 β, IL -2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, TNF-α, TNF-β and GM-CSF. For example, when the accumulation of immunocompetent cells around the site of administration of the immunopotentiating composition and the subsequent increase in antibody production is desired, IL-1β and IL-2 are particularly preferred.

Specific adjuvants of interest include, but are not limited to, one or more of the selected group of Adju-Phos, algal glucans, algamulin, alhydrogel, antigen formulation, avridine, Bay R1005, calcitriol, calcium phosphate gel, holotoxin. of cholera (CT), subunit B of cholera toxin (CTB), CRL1005, DDA, DHEA, DMPC, DMPG, DOC / alum complex, gamma inulin, Gerbu adjuvant, GMDP, Imiquimod, ImmTher, Interferon-gamma, ISCOM (s), Iscoprep 7.0.3, Loxoribine, oral adjuvant LT-OA or LT, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPL, MTP-PE, MTP-PE liposomes, Murametlda, Murapalmitin, D-Murapalmitin, NAGO , non-ionic surfactant vesicles, Pleuran, PLGA, PGA and PLA, Pluronic L121, PMMA, PODDS, Poly Ra: Poly rU, Polyphosphazene, Polysorbate 80, protein cochleates, QS-21, Quil A, Rehydragel HPA, Rehydragel LV , S-28463, SAF-1, Sclavo peptide, Sendai proteoliposomes, lipid matrices containing Sendai, Span 85, Specol, squalene, squalane, stearyl tyrosine, Teramide, Treonil-MDP, and Ty particles. The amount of the immuno-inducing antigenic substance and the immunomodulatory substance contained in the immunopotentiating composition of the present invention can be adjusted arbitrarily according to the mixing ratios for the biocompatible material and the additives contained in the composition, and for the shape or size of the composition. The dose of the antigenic substance that will be administered by the composition of the present invention may be approximately equal to that used for the conventional administration method (eg, parenteral administration in the form of a solution or suspension). However, since the composition of the present invention has an excellent immunopotentiating effect, as mentioned above, the immunity can be sufficiently induced at lower doses, in comparison with the conventional administration method, and the dose can be adjusted appropriately according to the antigenic substance, the dosage form of the composition of the present invention, and / or the immunomodulatory substance which will be administered simultaneously with the antigenic substance and the amount thereof. The form of the immunopotentiating composition of the present invention can be such that the composition is, for example, in the form of a solution, in the form of a suspension, in the form of a gel, in the form of a film, in the form of a sponge, in the form of a rod or bar, or in the form of a particle. A suitable form can be selected so that an immune response can be induced more efficiently. The rod shape is preferred for the composition. A coated or coated rod formulation, as described in EP 659,406, is more particularly preferred. For example, a stick-like shape can release the antigenic substance and, when present, the immunomodulatory substance for a prolonged period, while a particle-like composition can easily undergo phagocytosis by immunocompetent cells such as macrophages. In the case of fine particles or granules, the diameter thereof is conveniently, but not limited to, from 0.1 micrometers to 100 micrometers, more conveniently from 0.5 micrometers to 50 micrometers. The biocompatible carrier according to the present invention can be such that the antigenic substance is dispersed therein or encapsulated therein. The biocompatible carrier can be such as to allow delayed and / or sustained release of the antigenic substance. The biocompatible vehicle can be formed from any suitable biocompatible material. Preferred as the "biocompatible material" are those materials that have good biocompatibility and can retain the antigenic substance, or an antigenic substance and a immunomodulatory substance, stably and sustained release thereof in vivo. Thus, as a biocompatible material, there may be mentioned, for example, collagen, gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose, gum arabic.; polymers of glycolic acid, lactic acid or an amino acid, and copolymers of two or more thereof; hydroxyapatite, poly (methyl methacrylate), polydimethylsiloxane, polytetra-fluoroethylene, polypropylene, polyethylene, and mixtures of two or more of these biocompatible materials. The biocompatible material is selected to satisfy the condition that it must not denature and / or inactivate the antigenic substance, or the antigenic substance and the immunomodulating substance, in the process for preparing the immunopotentiating composition. It can be biodegradable (degradable in vivo) or non-biodegradable, depending on the desired effect. As particularly preferred biodegradable and biocompatible materials, collagen can be mentioned. It is also convenient that the collagen be used in combination with one or more other biocompatible materials. Any species of collagen can be used, so long as it is suitable for the purpose of the present invention. In this way, acid-soluble collagen derived from plants or animals, soluble collagen salts and alkali-soluble collagen derivatives thereof, derivatives thereof such as atheroocollagen, modified side-chain collagen and cross-linked collagen, and genetically engineered collagen can be used. , preferably atherosclergenic, modified side-chain collagen and cross-linked collagen. As side chain modified collagen, there may be mentioned, for example, succinylated, methylated or myristylated collagen. As entangled collagen, mention may be made, for example, of collagen treated with glutaraldehyde, hexamethylene diisocyanate or with polyepoxy compounds (Fragrance Journal, 1989 (12), 104-109, Japanese patent application (Kokoku) 07-59522). The polydimethylsiloxane can be mentioned as a particularly preferred non-biodegradable biocompatible material, and it is also convenient that one or more of the biocompatible materials mentioned above be used in admixture with this polydimethylsiloxane. Said polydimethylsiloxane is not limited to any particular species but, to facilitate the molding ability and other points of view, silicones are particularly preferred such as Q7-4750 elastomer Silastic (registered trademark) ETR commercial grade and MDX-4-4210 elastomer of Dow Corning (registered trademark) of medical grade. For the stabilization of the antigenic substance, or the antigenic substance and the immunomodulatory substance, and / or to control the release thereof, one or more pharmaceutical additives may be added. Pharmaceutical additives include, but are not limited to, albumin, glycine, amino acids other than glycine, polyamino acids, gelatin, chondroitin sulfate, sodium chloride, mannan, glucomannan, tannic acid, sodium citrate, mannitol, etc. In cases where one or more biocompatible materials or additives are used in mixtures with collagen, the proportion of collagen is preferably not less than 10% w / w, preferably on the scale of not less than 30% w / w, more preferably on the scale of not less than 70% in p / p. In cases where one or more other biocompatible materials or additives are used in mixtures with polydimethylsiloxane, the proportion of the polydimethylsiloxane is preferably not less than 10% w / w, preferably on the scale of not less than 50% w / w, more preferably on the scale of not less than 70% w / w.

As a biocompatible carrier, the combination of a collagen-based or silicone-based biocompatible carrier in the form of a rod or rod, preferably in the form of a covered rod, in combination with an active agent or immunomodulatory agent is preferred. Accordingly, in a preferred aspect of the present invention, an immunopotentiator is provided in solid unit dosage form that includes a biocompatible carrier formed of a biocompatible material based on silicone or collagen based; an antigenic substance, and an immunomodulatory substance carried therein. Preferably, the biocompatible carrier is in the form of a rod-shaped article, more preferably in the form of a covered rod. More preferably, the biocompatible rod-shaped carrier is formed of a silicone-based material. The inventors have found that, in this form, the antigenic substance can be stable at room temperature and thus does not require cold storage. In addition, the immunomodulatory agent can be introduced directly into the immunopotentiating composition; that is, without requiring solvent. The method for administering the immunopotentiating composition of the present invention is not particularly limited, but includes parenteral administration, oral administration, administration in the nasal cavity and / or lungs, discharge using compressed air, and retention or inclusion at the incision site. A suitable administration method can be selected for the form of the immunopotentiating composition, such that an immune response can be more effectively induced. In the case of rod-shaped or stick-like compositions, parenteral administration or retention at the incision site is desirable while, in the case of particles, they can be applied directly as such to the incision site for retention, or can be administered parenterally in the form of a prepared suspension, suspending them in a solvent for injection, as described in Japanese Patent Publication (Kokoku) 03-72046 or, in addition, they can be administered, for example, by means of compressed air discharge using the Helios system Gene Gun (Bio-Rad), or a spray gun described in Proc. Natl. Acad. Sci, USA, 93, 6291-6296 (1996). Although the solvent for injection must be selected depending on the properties of the biocompatible material, the antigenic substance and the immunomodulatory substance, the term "solvent for injection" is not limited to any particular species, provided that 1) the particle can be dispersed in the solvent, 2) the particle can maintain its shape when the particle is dispersed in the solvent, 3) the antigenic substance and the immunomodulatory substance can be maintained in the particle when the particle is dispersed in the solvent, 4) the solvent is not toxic, and 5) the solvent in which the particle is dispersed, is not toxic. For example, the solvent for injection, as referred to herein, includes, but is not limited to, distilled water, physiological saline solution, pH regulated solution with phosphate, soybean oil, sesame oil, peanut oil, oil cottonseed, MTC (medium chain fatty acid triglycerides) olive oil, corn oil, castor oil, silicone oils, PEG (polyethylene glycol), PG (propylene glycol) and fatty acids used in the preparation of liposomes, such as DOTMA, DOPE, DOGS, etc. As a method for producing the immunopotentiating compositions in the form of a biodegradable solution, suspension and aqueous gel, it can be mentioned, for example. (1) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of a powder, solution, suspension or gel, with a vehicle in the form of a solution or gel containing, if necessary, one or more additives, (2) the method that comprises allowing a solution, suspension or gel containing an antigenic substance, or an antigenic substance and an immunomodulatory substance, are added to a vehicle in the form of a powder containing, if necessary, one or more additives, (3) the method comprising allowing a solution, suspension or gel containing an antigenic substance, or an antigenic substance and an immunomodulatory substance, are added to a sponge-like vehicle containing, if necessary, one or more additives, followed by kneading. However, these are not limiting. The method for producing the immunopotentiating composition in the form of a biodegradable solid includes, but is not limited to, the method of Fujioka et al. (Japanese Patent Application Kokoku) 07-59522). As other methods, there may be mentioned: (1) The method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of powder, solution, suspension or gel, with a vehicle in the form of a solution or gel that contains, if necessary, one or more additives, followed by drying, (2) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of a powder, solution, suspension or gel, with vehicle in powder form containing, if necessary, one or more additives, followed by drying, (3) the method comprising allowing a solution, suspension or gel containing an antigenic substance, or an antigenic substance and an immunomodulating substance, to be added to a vehicle in the form of a sponge containing, if necessary, one or more additives, followed by drying, (4) the method comprising allowing a solution, suspension or gel containing an antigenic substance, or an antigenic substance and an immunomodulatory substance, be added to a sponge-like vehicle containing, if necessary, one or more additives, followed by direct drying or addition of water or its like or, if necessary, kneading and drying, (5) the method comprising grinding the solid obtained in methods (1) - (4), followed by compression molding, (6) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of a powder, with a vehicle in powder form containing, if necessary, one or more additives, followed by compression molding. The method for producing the immunopotentiating composition in the form of biodegradable particles includes, but is not limited to, (1) the method comprising spray drying a solution containing an antigenic substance, or an antigenic substance and a substance. The immunomodulator and the vehicle, if necessary, together with one or more additives, (2) the method comprising freeze-drying a solution containing an antigenic substance, or an antigenic substance and an immunomodulatory substance and the vehicle, if necessary, together with one or more additives, followed by milling the lyophilizate in the form of obtained sponge, and (3) the method comprising adding dropwise a solution containing an antigenic substance, or an antigenic substance and an immunomodulatory substance and the vehicle, if it is necessary, together with one or more additives, to a solution in which the vehicle is insoluble, and to dry the obtained particles. The drying method, the temperature and humidity in the drying step, the mixing method, the temperature and humidity in the mixing step, the compression molding method, the temperature, humidity and molding pressure in the molding step by compression, the viscosity of the solution of the vehicle and of the active substance, or the antigenic substance and the solution of immunomodulatory substance, and the viscosity and pH of the mixed solution of vehicle - antigenic substance and of the mixed solution of antigenic substance - immunomodulating substance, can be the same as conventional methods. The method for producing the immunopotentiating composition in the form of a non-biodegradable solid includes, but is not limited to, (1) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of a powder, solution, suspension or gel, with a carrier monomer with one or more additives added to it if necessary, adding a hardening agent, molding in an arbitrarily selected mold by filling or extrusion, and effecting hardening, (2) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of solution, suspension or gel, with a vehicle in powder form containing, if necessary, one or more additives, followed by drying, (3) the method comprising mixing an antigenic substance, or an antigenic substance and a substance. immunomodulator, in the form of powder, solution, suspension or gel, with a vehicle in powder form containing, if necessary, one or more additives, and (1) filling the in an arbitrarily selected mold, followed by compression molding, or (2) extruding the mixture using a nozzle, (4) the method comprising mixing an antigenic substance, or an antigenic substance and an immunomodulating substance, in the form of a solution, suspension or gel, with a vehicle in the form of a sponge containing, if necessary, one or more additives, followed by drying, (5) the method comprising mixing an active substance, or an active substance and immunomodulating substance, in the form of powder, solution, suspension or gel, with a vehicle in the form of a sponge containing, if necessary, one or more additives, and (1) filling the mixture in an arbitrarily selected mold, followed by compression molding, or (2) extruding the mixture using a nozzle, (6) the method comprising forming, by methods (1), (3) and (5), an inner layer in the form of a rod or bar containing an antigenic substance, or an antigenic substance and a sust the immunomodulator, and then coating the inner layer with an antigenic substance and an outer layer material free of immunomodulatory substance, and (7) the method comprising simultaneously forming an inner layer and an outer layer by coextrusion using a nozzle, between others. The drying method, the temperature and humidity in the drying step, the mixing method, the temperature and humidity in the mixing step, the compression molding method, the temperature, humidity and molding pressure in the molding step by compression, the viscosity of the vehicle solution and the antigenic substance, or the antigenic substance and the solution of immunomodulatory substance, and the viscosity and pH of the mixed solution of vehicle-antigenic substance and of the mixed solution of antigenic substance - Immunomodulatory substance, can be equal to those of conventional methods. The method of use of the immunopotentiating composition of the present invention includes, but is not limited to, for example (1) the use as a vaccine preparation for human use or for use in mammals other than humans and in birds, for the purpose of prevention or treatment of diseases, and (2) the use as an immunization preparation that will be administered to animals for the purpose of producing antibodies. Accordingly, in a preferred aspect of the present invention, there is provided a method for the prophylactic or therapeutic treatment of a disease or other disorder, which method includes providing an immunopotentiating composition that includes a biocompatible carrier.; and an antigenic substance, or an antigenic substance and an immunomodulatory substance carried therein; and administering to the subject an effective amount of the immunopotentiating composition. The site of administration of the immunopotentiating composition of the present invention can be selected in accordance with the purpose of use. In the case of use as an ordinary vaccine, for example, the composition can be administered subcutaneously, intramuscularly, or by a similar route. Since, as already mentioned under the "explanation of the principle of the present invention" section, the immunopotentiating composition of the present invention can specifically activate the immune response in the lymph node in charge of the administration site or in the vicinity of the site of administration. administration, said composition can be administered directly to a target organ, as necessary. Thus, for example, an immunopotentiating composition possessing an antigen derived from tumor and a cytokine, can be administered directly to loci of tumor cells or to a site from which a tumor has been removed by surgery, thus activating the immune response. to the tumor. Furthermore, it is expected that such direct local administration of the immunopotentiating composition to the tumor locus will produce an inhibitory effect on the metastasis of tumor cells to the systemic lymphatic system by the lymph node in charge of the tumor locus. The present invention will now be described in more detail with reference to the accompanying figures and examples. However, it should be understood that the following description is illustrative only, and that it should in no way be construed as a restriction on the generality of the invention described above.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 relates to the time courses of cumulative releases of avidin and IL-1β from immunopotentiating compositions, as described in test examples 1 and 2. Figure 2 refers to the time course of the title of Antiavidin antibody in mice. Figure 3 refers to the time course of the anti-avidin antibody titer in sheep. Figure 4 is a histological photomicrograph of the administration site of the immunopotentiating composition prepared in test example 7 (CD45, x300). Figure 5 is a histological microphotograph of the administration site of the immunopotentiating composition prepared in test example 8 (CD45, x300). Figure 6 refers to the time courses of the cumulative releases of avidin from the immunopotentiating compositions as described in test example 5. Figure 7 refers to the time courses of the cumulative releases of avidin and IL -1ß from the immunopotentiating compositions, as described in test example 6. 1 Figure 8 refers to the time courses of antiavidin antibody titers in mice, as described in test example 7. Figure 9 refers to the time courses of antiavidin antibody titers in mice, as shown in FIG. FIG. 10 describes the time course of the body temperature and the leukocyte count of sheep after the administration of silicone alone in the test example 9. FIG. 11 refers to the course of body temperature time and leukocyte count of sheep after administration of silicone-based immunopotentiating compositions containing IL-1β in test example 9. Figure 12 refers to the time course of body temperature and the leukocyte count of sheep after the administration of silicone-based immunopotentiating compositions containing IL-1β in test example 9.

EXAMPLES Preparation of immunopotentiating compositions EXAMPLE 1 An aqueous solution (10 ml) containing 5.0 mg / ml of avidin (Boehringer Mannheim GmbH, Germany) and 3 ml of an aqueous solution containing 100 mg / ml of glycine (Nakalai Tesque, Inc., Japan), were mixed with 134 g of a solution of atelocollagen (Koken Co., Ltd., Japan, atelocollagen content: 2%) to give an immunopotentiating composition in the form of a solution.

EXAMPLE 2 An aqueous solution (1.7 ml) containing 5.0 mg / ml sheep IL-1β (prepared by the method of AE Andrews et al., Vaccine, 12, 14-22, 1994), 8.6 ml of an aqueous solution containing 5.0 mg / ml of avidin and 1.5 ml of an aqueous solution containing 100 ml / ml of glycine, were mixed with 142 g of the atelocollagen solution at 2%, to give an immunopotentiating composition in solution form.

EXAMPLE 3 An immunopotentiating composition in the form of a sponge was obtained by lyophilizing the immunopotentiating composition in the form of the solution prepared in Example 1.

EXAMPLE 4 An immunopotentiating composition in the form of a sponge was obtained by lyophilizing the immunopotentiating composition in the form of the solution prepared in Example 2.

EXAMPLE 5 An immunopotentiating composition was obtained in the form of a gel by adding 7 g of distilled water to the immunopotentiating composition in the form of a sponge prepared in Example 3, allowing the mixture to stand overnight, followed by kneading.

EXAMPLE 6 An immunopotentiating composition was obtained in the form of a gel by adding 7 g of distilled water to the immunopotentiating composition in the form of a sponge prepared in Example 4, allowing the mixture to stand overnight, followed by kneading.

EXAMPLE 7 An immunopotentiating composition was obtained in the form of a rod, in the form of a rod, by extruding the immunopotentiating composition in gel form prepared in Example 5, followed by drying.

EXAMPLE 8 An immunopotentiary composition in the form of a rod, in the form of a rod, was obtained by extruding the immunopotentiating composition in gel form prepared in Example 6, followed by drying.

EXAMPLE 9 An aqueous solution of 1 mg / ml of avidin (11.1 g) and 12.2 g of an aqueous solution of 81 mg / ml of human serum albumin (HSA) are mixed together, and the mixture is lyophilized. The lyophilized grinds and sieves to give a powder with a particle size no larger than 20 micrometers. Separately, 0.7 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA), medical grade ETR, part A, and 0.7 g of part B are mixed together. After mixing, the mixture is rapidly kneaded with 0.6 g of the powder mentioned above. The kneaded mixture is extruded under pressure through a hole having a diameter of 1.9 mm and allowed to stand at room temperature for curing. The rod is cut to give an immunopotentiating composition.

EXAMPLE 10 An aqueous solution of 1 mg / ml of avidin (11.1 g), 61 microliters of an aqueous solution of 2 mg / ml of IL-1β solution and 12.2 g of an aqueous solution of 81 mg / ml of human serum albumin (HSA) are mixed, and the mixture is lyophilized. The lyophilized grinds and sieves to give a powder with a particle size no larger than 20 micrometers. Separately, 0.7 g of Q7-4750 Silastic elastomer (registered trademark, Dow Corning Co., USA), medical grade ETR, part A, and 0.7 g of part B are mixed together. After mixing, the mixture is rapidly kneaded with 0.6 g of the powder mentioned above. The kneaded mixture is extruded under pressure through a hole having a diameter of 1.9 mm and allowed to stand at room temperature for curing. The rod is cut to give an immunopotentiating composition.

EXAMPLE 11 The cured product of Example 9 is provided with an outer layer (thickness: 0.2 mm) by immersion in a dispersion of elastomer Q7-4750 Silastic at 10% (registered trademark, Dow Corning Co., USA) medical grade ETR (mixed from 1: 1 part A and part B) in toluene, followed by drying. The rod is cut to give an immunopotentiating composition.

EXAMPLE 12 The cured product of example 10 is provided with an outer layer (thickness: 0.2 mm) by immersion in a dispersion of elastomer Q7-4750 Silastic at 10% (registered trademark, Dow Corning Co., USA) medical grade ETR (mixed from 1: 1 part A and part B) in toluene, followed by drying. The rod is cut to give an immunopotentiating composition.

EXAMPLE 13 An aqueous solution of 1 mg / ml of avidin (11.1 g) and 12.2 g of an aqueous solution of 81 mg / ml of HSA solution are mixed together, and the mixture is lyophilized. The lyophilizate is milled and sieved to give a powder with a particle size no greater than 20 micrometers. Separately, 1,372 g of Shin-Etsu silicone (registered trademark, Shin-Etsu Chemical Co. Ltd., Japan), KE68 (main material) and 28 mg of ShinEtsu silicone (registered trademark, Shin-Etsu Chemical) are mixed together. Co., Ltd, Japan), Cat-RC (healing agent). After mixing, the mixture is rapidly kneaded with 0.6 g of the powder mentioned above. The kneaded mixture is extruded under pressure through a hole having a diameter of 1.9 mm, and allowed to stand at room temperature for curing. The rod is cut to give an immunopotentiating composition.

EXAMPLE 14 An aqueous solution of 1 mg / ml of avidin (11.1 g), 61 ml of an aqueous solution of 2 mg / ml of IL-1β and 12.2 g of an aqueous solution of 81 mg / ml of HSA are mixed, and the mixture It is lyophilized. The lyophilizate is milled and sieved to give a powder with a particle size no greater than 20 micrometers. Separately, 1372 g of Shin-Etsu silicone (registered trademark, Shin-Etsu Chemical Co. Ltd., Japan), KE68 (main material) and 28 mg of Shin-Etsu silicone (registered trademark, Shin-) are mixed together. Etsu Chemical Co., Ltd, Japan), Cat-RC (healing agent). After mixing, the mixture is rapidly kneaded with 0.6 g of the powder mentioned above. The kneaded mixture is extruded under pressure through a hole having a diameter of 1.9 mm, and allowed to stand at room temperature for curing. The rod is cut to give an immunopotentiating composition.

EXAMPLE 15 The cured product of example 13 is provided with an outer layer (thickness: 0.2 mm) by immersion in a 10% Shin-Etsu silicone dispersion (98: 2 mixture of KE-68 and Cat-RC) in toluene, followed by of drying. The rod is cut to give an immunopotentiating composition.

EXAMPLE 16 The cured product of Example 14 is provided with an outer layer (thickness: 0.2 mm) by immersion in a 10% Shin-Etsu silicone dispersion (98: 2 mixture of KE-68 and Cat-RC) in toluene, followed by of drying. The rod is cut to give an immunopotentiating composition.

EXAMPLE 17 An aqueous solution (0.578 g) containing 5 mg / ml of avidin, an aqueous solution (13.0 g) containing 100 mg / ml of sodium citrate and an aqueous solution (13.0 g) containing 100 mg are mixed and freeze-dried. / ml of mannitol. The lyophilized product was milled under a nitrogen atmosphere to provide a powder. Separately, 1.05 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA) medical grade ETR, part A, was mixed with 1.05 g of part B. After mixing, the mixture was quickly kneaded with 0.90 g of the previous powder. The kneaded mixture was filled in a syringe and extruded under pressure through the 1.6 mm hole, and allowed to stand at 25 ° C for 3 days for healing. The rod was cut to give an immunopotentiating composition.

EXAMPLE 18 An aqueous solution (2.89 g) containing 5 mg / ml of avidin, an aqueous solution (6.42 g) containing 100 mg / ml of sodium citrate and an aqueous solution (6.42 g) containing 100 mg are mixed and lyophilized. / ml of mannitol. The lyophilized product was milled under a nitrogen atmosphere to provide a powder. Separately, 0.93 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA), medical grade ETR, part A, was mixed with 0.93 g of part B. After mixing, the mixture was quickly kneaded with 0.80 g of the previous powder. The kneaded mixture was filled in a syringe and extruded under pressure through the 1.6 mm hole, and allowed to stand at 25 ° C for 3 days for healing. The rod was cut to give an immunopotentiating composition.

EXAMPLE 19 A kneaded mixture of avidin, sodium citrate, mannitol and Silastic was filled in a syringe in the same manner as in example 18. Separately, 50 g of Q7-4750 Silastic elastomer (trademark) were mixed and filled in another syringe. registered, Dow Corning Co., USA) medical grade ETR, part A, and 50 g of part B. Additions were extruded under pressure through concentrically arranged nozzles (outermost diameter: 1.9 mm), so that the Silastic that contained drug formed the inner part, and the drug-free Silastic, the outer part. The molded material was allowed to stand at 37 ° C for 5 days for healing, and was then cut to give an immunopotentiating composition.

EXAMPLE 20 An aqueous solution (0.30 g) containing 5 mg / ml of avidin, an aqueous solution (4.34 g) containing 100 mg / ml of sodium citrate and an aqueous solution (8.67 g) containing 100 mg are mixed and lyophilized. / ml of mannitol. The lyophilized product was milled under a nitrogen atmosphere to provide a powder. Separately, 0.93 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA), medical grade ETR, part A, was mixed with 0.93 g of part B. After mixing, the mixture was quickly kneaded with 0.80 g of the previous powder. The 5 The kneaded mixture was filled in a syringe and extruded under pressure through the 1.6 mm hole, and allowed to stand at 25 ° C for 3 days for healing. The molded material was cut to give an immunopotentiating composition.

EXAMPLE 21 A kneaded mixture of avidin, sodium citrate, mannitol and Silastic was filled into a syringe in the same manner as in example 20. Separately, 50 g of Q7-4750 Silastic elastomer (trademark) were mixed and filled in another syringe. Registered, Dow Corning Co., USA) medical grade ETR, part A, and 50 g of part B. Additions were extruded under pressure through concentrically arranged nozzles (inner diameter of an outer part: 1.9 mm, inner diameter of a inner part: 1.6 mm), so that the drug-containing Silastic formed the inner part, and the drug-free Silastic formed the outer part. The molded material was allowed to stand at 25 ° C for 5 days for healing, and then cut to give an immunopotentiating composition.

EXAMPLE 22 An aqueous solution (0.45 g) containing 5 mg / ml of avidin, an aqueous solution (3.15 g) containing 2 mg / ml of IL-1β, an aqueous solution (1.92 g) containing 250 mg were mixed and lyophilized. / ml of sodium citrate and an aqueous solution (6.19 g) containing 150 mg / ml of mannitol. The lyophilized product was milled under a nitrogen atmosphere to provide a powder. Separately, 1.05 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA) medical grade ETR, part A, was mixed with 1.05 g of part B. After mixing, the mixture was quickly kneaded with 0.90 g of the previous powder. The kneaded mixture was filled in a syringe and extruded under pressure through the 1.6 mm hole, and allowed to stand at 25 ° C for 5 days for healing. The molded material was cut to give an immunopotentiating composition.

EXAMPLE 23 A kneaded mixture of avidin, IL-1β, sodium citrate, mannitol and Silastic was filled in a syringe in the same manner as in example 22. Separately, 50 g of Q7-4750 elastomer Silastic (registered trademark, Dow Corning Co., USA) medical grade ETR part A, and 50 g of part B, were mixed and filled in another syringe. Additions were extruded under pressure through concentrically arranged nozzles (inner diameter of an outer part: 1.9 mm, internal diameter of an inner part: 1.6 mm), so that the Silastic containing drug formed the inner part, and the Silastic drug free, the outside. The molded material was allowed to stand at 25 ° C for 3 days for healing, and was then cut to give an immunopotentiating composition.

Release tests EXAMPLE OF TEST 1 The immunopotentiating composition prepared in Example 7 (10 mg) was placed in 5 ml of pH phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin and 0.01% sodium azide, and the avidin released was set tested by enzyme-linked immunosorbent assay (ELISA), and cumulative release was determined. The results thus obtained are shown in table 1. The immunopotentiating composition liberated avidin continuously for not less than 7 days.

EXAMPLE OF TRIAL 2 The immunopotentiating composition prepared in Example 8 (10 mg) was placed in 5 ml of pH phosphate buffer (pH 7.4) containing 0.5% bovine serum albumin and 0.01% sodium azide, and avidin and IL- 1β released were tested by ELISA test, and cumulative releases were determined. The results obtained are shown in table 1. The immunopotentiating composition released avidin and IL-1β sustained for not less than 7 days.

Antibody production experiment EXAMPLE OF TEST 3 Two groups of 5 female Balb / C mice received a subcutaneous administration of the immunopotentiating composition prepared in Example 7 containing 100 micrograms of avidin, the immunopotentiating composition prepared in Example 8 containing 100 micrograms of avidin and 20 micrograms of IL-1β. Blood samples were obtained from the mice on days 7, 14, 21, 35 and 83 after administration. Equal amounts of serum from the 5 mice in each group were pooled and tested for antiavidin-specific antibody by ELISA test. The results are expressed as midpoint titers at 50%, and demonstrate the immunopotentiating effects of the compositions (Figure 2). At 35 days after the administration, the antibody titer in the blood of the mice to which the immunopotentiating composition prepared in Example 7 was administered was approximately 25 times higher, in comparison with the antibody titer obtained in the CONTROL EXAMPLE 1. In addition, the antibody titer in the blood of the mice administered the immunopotentiating composition prepared in Example 8 was as high as approximately 450 times the antibody titer obtained in control example 1. .

EXAMPLE OF CONTROL 1 female Balb / C mice received a subcutaneous administration of 100 micrograms of avidin soluble in PBS. Blood samples were obtained from the mice on days 7, 14, 21, 35 and 83 after administration. Equal amounts of serum from the five mice were pooled and tested for antiavidin-specific antibody by ELISA test. The results are expressed as midpoint titers at 50%, and demonstrate the immunopotentiating effects of the compositions (Figure 2).

EXAMPLE OF TEST 4 merino sheep from mixed sex received a subcutaneous administration of the immunopotentiating composition produced in example 8. Blood samples were obtained from the lambs on days 7, 14, 21 and 35 after administration. Equal amounts of serum from the 5 lambs were pooled and tested for antiavidin-specific antibody by ELISA test. The results are expressed as midpoint titers at 50%, and demonstrate the immunopotentiating effects of the compositions (Figure 3). A high level of production of antiavidin-specific antibody was determined at 14 days after administration.

EXAMPLE OF CONTROL 2 Five merino sheep from mixed sex received a subcutaneous administration of 100 micrograms of avidin soluble in PBS. Blood samples were obtained from the lambs on days 7, 14, 21 and 35 after administration. Equal amounts of serum from the five lambs were pooled and tested for antiavidin specific antibody by ELISA test. The results are expressed as a mid-point titer at 50%, and demonstrate the immunopotentiating effects of the compositions (Figure 3). No production of antiavidin-specific antibodies was observed around 35 days after administration.

EXAMPLE OF CONTROL 3 Five merino sheep from mixed sex received a subcutaneous administration of 100 micrograms of soluble avidin and 20 micrograms of IL-1β in PBS. Blood samples were obtained from the lambs on days 7, 14, 21 and 35 after administration. Equal quantities of serum from the five sheep were pooled and tested for antiavidin-specific antibody by ELISA test. The results are expressed as midpoint titers at 50%, and demonstrate the immunopotentiating effects of the compositions (Figure 3). No production of antiavidin-specific antibodies was observed around 35 days after administration.

Histological analysis The sheep received a subcutaneous administration of the immunopotentiating composition prepared in Example 7 or the immunopotentiating composition prepared in Example 8, at three different sites on the left flank. The animals were sacrificed, and skin biopsies were taken for analysis at 72 hours after administration. Biopsies were included in OCT for analysis of cell surface CD45 expression on frozen sections. The histological micrograph demonstrates the leukocyte infiltration induced by the immunopotentiating compositions (Figures 4 and 5).

EXAMPLE OF TEST 5 The immunopotentiating composition prepared in Example 17 and cut to a size corresponding to an avidin content of 10 micrograms, and the immunopotentiating compositions prepared in Examples 18 and 19 and each cut to a size corresponding to an avidin content of 100 micrograms, were placed respectively in 2 ml of phosphate pH regulator (pH 7.4) containing 0.3% of Tween 20 and 0.01% of sodium azide, and allowed to stand. The released avidin was tested by ELISA, and the cumulative release was determined. The results obtained are shown in Figure 6. The kinetics of avidin release could be controlled by selecting the shape of the composition. In this way, the matrix-like compositions (examples 17 and 18) showed a nearly first order release pattern, while the coated rod-shaped composition (example 19) showed a nearly zero order release pattern. These compositions released avidin consistently for at least 30 days.

EXAMPLE OF TRIAL 6 The immunopotentiating compositions prepared in Examples 20 and 21 and each cut to a size corresponding to an avidin content of 5 micrograms, and immunopotentiating compositions prepared in Examples 22 and 23 and each cut to a size corresponding to a Avidin content of 5 micrograms and a content of IL-1β of 5 micrograms, were respectively placed in 2 ml of phosphate pH regulator (pH 7.4) containing 0.3% Tween 20 and 0.01% sodium azide, and They let it rest. Released avidin and IL-1β were tested by ELISA, and cumulative releases were determined. The results obtained are shown in Figure 7. As in Test Example 5, the kinetics of release of avidin and IL-1β can be controlled by selecting the form of the composition. These compositions released avidin and IL-1β sustained for at least 15 days.

EXAMPLE OF TEST 7 Three groups of Balb / C mice (six males per group) received subcutaneous administration of the immunopotentiating composition prepared in Example 17 (containing 10 micrograms of avidin), the composition prepared in Example 18 (containing 100 micrograms of avidin) and the composition prepared in Example 19 (containing 100 micrograms of avidin), respectively. Blood samples were obtained at 14, 28 and 42 days after administration. At each collection time, serum aliquots of the six mice in each group were pooled and tested for antiavidin antibody titer by ELISA test. Each antibody titer was expressed in the midpoint titer at 50%. The results obtained in this way are shown in Figure 8. At 14 days after the administration, the antibody titer in the blood of the mice to which the immunopotentiating composition prepared in Example 17 was administered, reached a level almost 180 times as high as that obtained in control example 4, despite the fact that the amount of avidin was only one tenth of the amount of the composition of control example 4. At 14 days after administration, the titers antibody in blood of the mice to which the immunopotentiating compositions prepared in Examples 18 and 19 were administered, were approximately 250 and approximately 190 times as high as those of Control Example 4. The antibody titers in the blood of the mice to which the immunopotentiating compositions prepared in examples 17, 18 and 19 were administered were greater than those in the control example. 4 for 6 weeks after administration.

EXAMPLE OF CONTROL 4 Balb / C mice (6 males) were subcutaneously administered a PBS solution containing 100 micrograms of avidin. At 14, 28 and 42 days after the administration, blood samples were obtained. At each collection time, aliquots of sera from the six mice were pooled and tested for antiavidin antibody titers by ELISA test. Each antibody titer was expressed in the midpoint titer at 50%. The results obtained are shown in table 8.

EXAMPLE OF TEST 8 Four groups of Balb / C mice (six males per group) received subcutaneous administration of the immunopotentiating composition prepared in Example 20 (containing 5 micrograms of avidin), the composition prepared in Example 21 (containing 5 micrograms of avidin), the composition prepared in example 22 (containing 5 micrograms of avidin and 5 micrograms of IL-1β), and the composition prepared in example 23 (containing 5 micrograms of avidin and 5 micrograms of IL-1β), respectively. Blood samples were obtained at 14, 28 and 42 days after administration. In each batch time, serum aliquots of the six mice in each group were pooled and tested for antiavidin antibody titer by ELISA test. Each antibody titer was expressed in the midpoint titer at 50%. The results thus obtained are shown in FIG. 9. In the mice to which the immunopotentiating compositions prepared in the examples were administered., 21, 22 and 23, the antiavidin antibody was depleted in blood starting 14 days after administration, while the antibody titer in blood in mice given the same amount of avidin in the example of control 5, was lower than the detection limit throughout the test period. The antibody titers in the blood of the mice to which the immunopotentiating compositions of Examples 20, 22 and 23 were administered were higher than those of Control Example 6 up to 28 days after administration, and the antibody titers In the blood of the mice to which the immunopotentiating compositions of Examples 22 and 23 were administered, they were much larger than those of Control Example 5 throughout the test period.

EXAMPLE OF CONTROL 5 Balb / C mice (six males) were subcutaneously administered a PBS solution containing 5 micrograms of avidin. At 14, 28 and 42 days after the administration, blood samples were obtained. At each collection time, aliquots of sera from the six mice were pooled and tested for antiavidin antibody titer by ELISA test. Each antibody titer was expressed in the midpoint titer at 50%. The results obtained are shown in Figure 9.

EXAMPLE OF CONTROL 6 Balb / C mice (six males) were subcutaneously administered a solution of PBS containing 5 micrograms of avidin and 0.26% by weight of alum. At 14, 28 and 42 days after the administration, blood samples were obtained. At each collection time, aliquots of sera from the six mice were pooled and tested for antiavidin antibody titer by ELISA test. Each antibody titer was expressed in the midpoint titer at 50%. The results are shown in Figure 9. In the sequential, the "immunopotentiating composition" is abbreviated as IC.

EXAMPLE OF TEST 9 A silicone-based IC was prepared in a manner similar to that described in examples 20 and 22 above. The silicon-based IC is identified as "matrix" in the following tables. The contents of each composition are shown in Table 1 below. In the same way, a silicone-based IC coated in a similar manner to that described in examples 21 and 23 above was prepared. The silicon-coated IC is identified as "covered rod" in the following tables. The contents of each composition are shown in Table 1 below.

TABLE 1 Contents of the compositions for the evaluation of the immunopotentiating effect of the silicone-based ICs, in the test example 9 In this test, immunopotentiating compositions based on silicone or control compositions (groups 10, 11 and 12) were introduced by the subcutaneous route, followed by a secondary immunization of 100 μg of avidin in PBS on day 28.

Efficacy of the silicone-based immunopotentiating composition The sheep were divided into 12 groups of 7 sheep per group.

Each group received a subcutaneous administration of a composition according to Table 1. A secondary immunization with 100 μg of avidin occurred on day 28. The results obtained are shown in table 2.

TABLE 2 Antiavidin antibody titers in sheep, as found in test example 9 (midpoint titers at 50%) M: CR matrix: covered rod S: PBS solution A: PBS solution containing alum.

The antiavidin specific antibody released was determined by ELISA test with titrations carried out from pooled serum of each group. In the absence of IL-1β adjuvant, the silicone IC of matrix was superior to the antigen delivered in saline (at all doses tested), but less effective than the antigen supplied in alum. The antibody response was increased by the addition of IL-1β as an adjuvant in silicone and saline compositions. Several ICs of silicone with IL-1β induced superior avidin responses in the alum composition. For the silicon IC of matrix, there was a tendency towards the lower dose of antigen, being more efficient for the induction of high antibody responses. The coated rod IC was greater than the matrix IC, based on the antibody titer and the duration of the response.

Biocompatibility of the silicon IC The sheep were divided into 8 groups of 7 sheep per group. Each group received a subcutaneous administration of a HF in accordance with Table 3.

TABLE 3 Contents of the silicone-based ICs for the evaluation of biocompatibility in test example 9 Two sheep were monitored per group for leukocyte count and body temperature. Biopsies were taken from the implant sites for histology at: 2 days after implantation (2 sheep) 4 weeks after implantation (2 sheep) 8 weeks after implantation (2 sheep) Two days after implantation, moderate edema was observed at the site of all ICs. This was more obvious in ICs with IL-1β. At 4 and 8 weeks after implantation, the tissues surrounding the IC sites appeared normal. The ICs were not encapsulated and were not adhering to the tissues, but they could move freely. No adverse reactions were observed in the tissues.

Systemic effects Leukocyte counts and body temperature measurements were carried out. The results obtained are shown for the silicone IC only in Figures 10a and 10b, for ICs that incorporate only IL-1β in Figures 11a and 11b, and for ICs with avidin and IL-1β in Figures 12a and b . All types of silicone implants that did not incorporate IL-1β did not induce any adverse effect on measurements of body temperature or leukocyte count. When silicone IC incorporating only IL-1β was administered, transient increases in body temperature were observed in the sheep, which were less severe than the results observed after the injection of IL-1β in saline. The average increase in these sheep was 1 ° C, and normal temperatures were observed for 24 hours. In addition, leukocyte counts increased up to 4 times more than normal levels; however, normal levels were observed again after 24 hours. The presence of avidin in the silicone IC resulted in a reduction in the severity and persistence of leukocyte counts (WBC) and temperature changes associated with the release of IL-1β from the IC. These results demonstrate that silicone ICs do not induce long-term aberrations in body temperature and leukocyte counts, and that the transient fluctuations that were observed were minimal and related to the inclusion of IL-1β in the ICs, more than any activity of the silicon ICs themselves.

Immunohistological evaluation of the administration sites The results for the different types of silicone ICs were identical; the different responses were attributable only to the presence or absence of IL-1β in the ICs. 1. ICs that incorporate IL-1ß. Day 2: In all animals that received ICs that contained IL-1β, there was a massive influx of cells, mainly neutrophils, around the site of administration and throughout the surrounding tissue. The cells stained for T and B cell markers were mainly in the epidermis, and only some of them were scattered in the lower layers of the skin. Week 4: The immune cells observed in these sections were mainly neutrophils. An increase in MHC class I and II positive cells above the level observed on day 2 was evident. A few CD4, CD8,? and d and positive CD1 occurred in the layer around the IC, and scattered through the rest of the tissue. Some positive CD45R cells also appeared scattered through the skin. Week 8: The cells were now mainly as a layer around the CI, and the tissue now had a more normal appearance.

The ACL-positive cells surrounded the ICs, and a layer of cells that did not stain with any of the lymphocyte markers was also evident. It is possible that these cells were fibroblasts. Paraffin sections stained with Masson's trichome showed a thin layer of densely stained collagen, indicating the start of encapsulation of the CI. MHC class I and II positive cells also occurred in the positive ACL layer around the IC, but not along the tissue as in week 4. There are only scattered positive cells for CD4, CD8,?, D, CD1, and CD45R. 2. ICs sín lL-1 ß. Day 2: Biopsies taken from animals that received ICs that did not include IL-1ß had the appearance of normal skin. No cell influx or edema was observed. Minimal surface marker staining of lymphocytes occurred in cells of the epidermis. Week 4: Fibroblasts were evident around the administration sites and, in addition, the IC of the covered rod type showed cells Positive LCA in the open end of the CI. These cells were mainly MHC class I positive cells with a few class II cells and CD4 positive Week 8: There were some fibroblasts around the CI, and cells that were stained with any of the lymphocyte surface markers were rare. These results indicate that the CIs of sílicón were well tolerated after the subcutaneous administration in sheep, and that adverse reactions were not observed that limited their use after 8 weeks of administration.

EXAMPLE OF TEST 10 Test example 9 was repeated using a collagen-based IC, which was prepared in a manner similar to that described in examples 7 and 8. The composition of the CI is shown in table 4 below, and the results obtained were show in table 5 below.

TABLE 4 Contents of the compositions for the determination of the dependence of the immunopotentiating effect of collagen-based ICs on the amounts of antigen and cytokine in the test example 10 * Subcutaneous use Secondary immunization with 100 μg of avidin in PBS.

TABLE 5 Dependence of the immunopotentiating effect of HCA based on collagen on the amounts of antigen and cytokine in Test Example 10 (medium point titer at 50%) The results confirm that the inclusion of IL-1β in the collagen IC increases the antibody response to avidin. The optimal dose of IL-1β could not be established statistically; however, the highest response was recorded when the two highest doses of IL-1β were administered. The results obtained were compared with the HF of collagen with alum and PBS (see table 6).

TABLE 6 Contents of the compositions for the evaluation of the immunopotentiating effect of HCA based on collagen, in test example 10 i.m .: intramuscular administration The subcutaneous route was used, except when indicated, and secondary administration was carried out with 100 μg of avidin in PBS. 28 days after the secondary immunization, delayed-type hypersensitivity responses were examined by intradermally injecting 1 μg of avidin in PBS into the wool-free region of the inner thigh of the sheep. The site was examined 24 and 48 hours after the injection for the detection of edema and erythema. The results obtained are shown in tables 7 and 8.

TABLE 7 Anti-avidin antibody titers in sheep, as found in test example 10 (mid-point titers at 50%) TABLE 8 Score of delayed-type hypersensitivity in test example 10 7 E: erythema O: edema Collagen IC did not induce any reaction of strong delayed-type hypersensitivity.

Moderate DTH reactions were recorded for sheep immunized with liquid compositions. No immediate hypersensitivity reactions were observed in any animal.

EXAMPLE OF TEST 11 The test examples 9 and 10 were repeated to evaluate the effects of individual dose immunization using IC as specified in table 9. The results obtained are shown in tables 10 and 11.

TABLE 9 Contents of the compositions for the evaluation of the immunopotentiating effect with individual dose immunization in the test example 11 * Subcutaneous use TABLE 10 Titers of antiavidin antibody in sheep, as found in test example 11 (mid-point titers to 50%) Cl: Collagen IC M: IC of matrix A: PBS solution containing alum S: PBS solution CR: Coated rod IC TABLE 11 Score of delayed-type hypersensitivity in test example 11 The collagen and silicone IC did not induce any reaction of strong hypersensitivity of delayed type. Moderate DTH reactions were recorded for sheep immunized with liquid compositions.

No immediate hypersensitivity reactions were observed in any animal. The silicone IC of coated rod was the most effective formulation for immunization of individual doses, inducing the highest titers and the most persistent response. The effective use of the coated rod IC for immunization was dependent on the inclusion of IL-1 3. The coated rod IC did not inherently induce delayed-type hypersensitivity responses. An effective memory response was induced in animals that received ICs were incorporated IL-1/3. This is indicated by the response after the secondary immunization was administered on day 69, after the titles for the first immunization had declined. The analysis of isotype determination in these serum samples was also carried out. The results indicate high levels of IgG when present throughout the course of the experiment in all groups, while IgM was detectable at low levels only at 14 days. This indicates that the isotype change had effectively occurred only after a single administration of antigen.

EXAMPLE OF TEST 12 Test example 11 was repeated using a series of antigens which can prevent infection by disease, instead of the model avidin antigen, as specified in table 12.

TABLE 12 Contents of the compositions for the evaluation of the inimunopotentiating effect with clostridial antigens, in the test example 12 TABLE 13 Antibody titers for anti-clostridial antigen in sheep, as found in test example 12 (mid-point titers at 50%) The results obtained are shown in Table 13. The coated rod IC is clearly superior to the conventional alum vaccine and the combination of alum / IL-1β for both antigens tested. Collagen IC induced titers that were not significantly different from the alum formulation; however, at later time points, the rod IC covered with toxoid novyi induced titers that were more than 2 times higher than for the alum formulation.

EXAMPLE OF TEST 13 A dose response analysis was performed using silicon-based IC. The composition and structure of each IC are shown in Table 14 below. Antibody titers were carried out on a biweekly basis. The results obtained are shown in table 15.

TABLE 14 Contents of the compositions for the determination of the dependence of the immunopotentiating effect of the silicone-based ICs on the amounts of antigen and cytokine in the test example 13 TABLE 15 Dependence of the immunopotentiating effect of silicone-based ICs on the amount of antigen and cytokine in test example 13 (mid-point titers at 50%) Cl: Collagen IC M: IC of matrix A: PBS solution containing alum S: PBS solution CR: Coated rod IC.

These results demonstrate the high titer and the persistence of antibodies that occur in response to the coated rod ICs. The most persistent response was induced in the presence of the highest dose of IL-1β and antigen. At 70 days, the coated silicone ICs incorporating IL-1β were clearly superior to the liquid avidin formulations (titers of approximately 5 times). The collagen and silicone IC could function effectively as a vaccine vehicle: the immunogenicity of the antigen was retained, and the biological activity of the cytokine adjuvant was preserved. Collagen IC and silicone matrix IC exhibited inherent adjuvant activity. When IL-1β was incorporated into the IC at appropriate levels, the antibody responses exceeded the responses induced by the alum adjuvant. The coated rod silicone IC incorporating IL-1β as an adjuvant induced significantly higher antibody responses than any other composition tested (liquid or IC). In addition, the antibody response was sustained for longer periods than for other compositions. No adverse systemic or histological responses have been observed that would exclude the use of CIs as vehicles for safe vaccines. Finally, it will be understood that various other modifications and / or alterations may be made without departing from the spirit of the present invention as described herein.

Claims (32)

NOVELTY GIVE THE INVENTION CLAIMS

1. - An immunopotentiating composition, characterized in that it comprises an antigen or antigen-inducing substance, and a vehicle comprising collagen or polydimethylsiloxane.

2. The immunopotentiating composition according to claim 1, further characterized in that it comprises at least one pharmaceutical additive.

3. The immunopotentiating composition according to claim 1 or 2, further characterized in that the antigen or antigen-inducing substance is capable of inducing a specific immune response to a member of the group consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells.

4. The immunopotentiating composition according to claim 3, further characterized in that the antigen or antigen-inducing substance is a substance obtained by the use of chemical technology, recombinant DNA technology, cell culture technology or fermentation technology.

5. The immunopotentiating composition according to claim 3, further characterized in that the antigen is derived from a member consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells, by attenuation, detoxification, or by making them the same. no pathogens.

6. The immunopotentiating composition according to claim 3, further characterized in that the antigen or antigen-producing substance is a substance obtained from a member of the group consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells.

7. The immunopotentiating composition according to claim 3, further characterized in that the antigen-producing substance is a plasmid or virus with a nucleic acid (gene sequence) that codes for an antigen capable of inducing a specific immune response to a member of the group consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells as incorporated herein, so that said antigen can be produced live.

8. The immunopotentiating composition according to claim 1 or 2, further characterized in that said composition is a solution, suspension, gel, film, sponge, rod or bar, or minute particles.

9. The immunopotentiating composition comprising an antigen or antigen-inducing substance, a substance having immunoactivation activity, immunostimulation or immunomodulation, and a vehicle comprising collagen or polydimethylsiloxane.

10. The immunopotentiating composition according to claim 9, further characterized in that the substance having immunoactivation, immunostimulation or immunomodulation activity is a cytokine.

11. The immunopotentiating composition according to claim 9 or 10, further characterized in that it comprises at least one pharmaceutical additive.

12. The immunopotentiating composition according to any of claims 9 to 11, further characterized in that the antigen or antigen-inducing substance is capable of inducing a specific immune response to a member of the group consisting of viruses, mycoplasmas, bacteria, parasites. , toxins and tumor cells.

13. The immunopotentiating composition according to claim 12, further characterized in that the antigen or antigen-inducing substance is a substance obtained by the use of chemical technology, recombinant DNA technology, cell culture technology or fermentation technology.

14. The immunopotentiating composition according to claim 12, further characterized in that the antigen is derived from a member consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells, by attenuation, detoxification, or by making them the same. no pathogens.

15. - The immunopotentiating composition according to claim 12, further characterized in that the antigen or antigen-inducing substance is a substance obtained from a member of the group consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells.

16. The immunopotentiating composition according to claim 12, further characterized in that the antigen-inducing substance is a plasmid or virus with a nucleic acid (gene sequence) that codes for an antigen capable of inducing a specific immune response to a member from the group consisting of viruses, mycoplasmas, bacteria, parasites, toxins and tumor cells as incorporated herein, so that said antigen can be produced in vivo.

17. The immunopotentiating composition according to any of claims 9 to 11, further characterized in that said composition is a solution, suspension, gel, film, sponge, rod or bar, or minute particles.

18. The immunopotentiating composition according to claim 1 or 2, further characterized in that the antigen is a superantigen.

19. The immunopotentiating composition according to claim 1 or 2, further characterized in that the antigen-inducing substance is a plasmid or virus with a nucleic acid (gene sequence) encoding superantigen as incorporated herein, so that said superantigen can be produced in vivo.

20. The immunopotentiating composition according to any of claims 9 to 11, further characterized in that the antigen is a superantigen.

21. The immunopotentiating composition according to any of claims 9 to 11, further characterized in that the antigen-producing substance is a plasmid or virus with a nucleic acid. (gene sequence) coding for superantigen as incorporated herein, so that said superantigen can be produced in vivo.

22. The immunopotentiating composition according to claim 1 or 9, further characterized in that said composition has the form of a rod or rod.

23. The immunopotentiating composition according to claim 22, further characterized in that said composition has the form of a coated or coated rod.

24. The immunopotentiating composition according to claim 23, further characterized in that the vehicle is a polydimethylsiloxane.

25. The immunopotentiating composition according to claim 22, further characterized in that during use, it exhibits a modified release profile.

26. The immunopotentiating composition according to claim 25, further characterized in that during use, it exhibits a sustained release profile.

27. The immunopotentiating composition according to claim 9, further characterized in that the substance having immunoactivation, immunostimulation or immunomodulation activity is an adjuvant selected from the group consisting of cytokines, chemokines, growth factors, DNA and peptide sequences. adjuvants, alum, complete Freund's adjuvant, Freud's incomplete adjuvant, iscom, saponins, hexadecylamine, dimethyldioctadecylammonium bromide, Abridin, skeletal components of the cell wall, cholera toxin, lipopolysaccharide endotoxins, and liposomes.

28. The immunopotentiating composition according to claim 10, further characterized in that the cytokine is included in the composition in the absence of a solvent.

29. A method for producing an antibody, characterized in that it comprises administering the immunopotentiating composition of any of claims 1 to 28 to a human being, to a mammal other than a human being or to a bird, thereby modulating the immune response in said mammal or bird, and recovering the antibody produced.

30. A method for the prophylactic or therapeutic treatment of a disease or other disorder, characterized in that said method includes: providing an immunopotentiating composition comprising collagen or polydimethylsiloxane, an antigen or antigen-producing substance; and administering to a subject an effective amount of the immunopotentiating composition.

31. The method according to claim 30, further characterized in that the immunopotentiating composition further includes an immunomodulation agent.

32. The method according to claim 31, further characterized in that the subject is a human being, a mammal different from a human being, or a bird.