Inorganic nanoparticle-based vaccine compositions for cancer treatment

Abstract

The present invention relates to the field of biotechnology, particularly human health. The present invention provides new vaccine compositions comprising as active principles systems containing recombinant human epidermal growth factor (EGF) or peptides thereof and carrier proteins or peptides, bound to a core composed of inorganic nanoparticles with nanometer or submicrometer dimensions. These vaccine compositions are suitable for use in the chronic treatment of cancer and have the advantage of not causing side effects at the injection site and not accumulating in the body.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, particularly human health, and in particular describes a new vaccine formulation that comprises a nanoparticulate system that induces an immune response against epidermal growth factor and is therefore useful as a cancer therapeutic. [Background technology]

[0002] Tumors of epithelial origin generally express receptors for epidermal growth factor (EGF) on their surface. Binding of self-EGF to its receptor (EGFR) activates tumor mechanisms that promote tumor proliferation and evade immune system defenses. Based on this finding, since the 1990s, EGF has been identified as a therapeutic target for lung cancer and other cancers of epithelial origin, and the binding of EGF to its receptor has been implicated.

[0003] Many studies have been conducted to evaluate passive immunotherapy using EGFR-targeting antibodies. Previously, it was shown that specific recognition of the receptor by antibodies of mouse origin inhibits the mitogenesis of malignant cells (Sato JD et al., Methods in Enzymology (1987) 146:63-81). Another study published in US 5,891,996 patent describes chimeric and humanized monoclonal antibodies that can play a diagnostic or therapeutic role in tumors with overexpression of EGFR.

[0004] The use of active immunotherapy has expanded rapidly in recent years due to its potential to fight cancer and turn it into a chronically manageable disease. Unlike passive therapies, active therapies are based on stimulating the immune system to act against tumors and other factors that promote their development. In particular, they have the advantage that only small doses of the active substance are required, reducing toxicity.

[0005] The formulation mentioned in US 5,894,018 patent claims a vaccine composition that induces an immune response against autologous EGF. It contains autologous EGF bound to a carrier protein (cholera toxin B subunit, tetanus toxoid, monoclonal antibody or a protein from the outer membrane of Neisseria meningitidis) adjuvanted with aluminum hydroxide. On the other hand, US 8,778,879 patent describes a vaccine composition for therapeutic use in cancer patients, which shows active component The present invention relates to a chemical conjugate of recombinant human EGF (rhEGF) and recombinant protein P64k. It describes a purification procedure for the chemical conjugate that improves the purity and immunogenic activity of the chemical conjugate. The composition includes an adjuvant, which may be aluminum hydroxide or Montanide.

[0006] Subsequent clinical trials demonstrated that the therapeutic vaccine derived from the aforementioned invention was safe and effective for the treatment of advanced stages of non-small cell lung cancer (NSCLC) (Rodriguez PCat al., Clinical Cancer Research (2016), 22(15): 3782-3790). However, patients who achieved a high survival rate (more than 5 years) proved to suffer from damage to the muscles at the injection site (deltoid and buttocks) as a result of the accumulation of mineral oil contained in the adjuvant. This eventually forced some patients to discontinue treatment or to inoculate muscles more distant from the lymphatic organs.

[0007] Immunological adjuvants arose from the need to increase the immunogenicity of vaccines developed from recombinant proteins. The first adjuvant used was the double sulfate of aluminum in 1926. Subsequently, various inorganic adjuvants were evaluated, including aluminum, calcium, magnesium, iron, zinc, zirconium, cerium, beryllium, and silicon. However, only aluminum and calcium salts have been approved for use in humans (Paneque-Quevedo A., Applied Biotechnology (2013) 30: 243-249). In all studies carried out to date, inorganic adjuvants were used in combination with heterologous antigens.

[0008] Aluminum hydroxide and aluminum phosphate adjuvants are not well suited for chronic treatment because aluminum is toxic and poorly metabolized in the human body. The effects of aluminum accumulation on human health are well documented, including the central nervous system, musculoskeletal system, respiratory system, circulatory system, endocrine system, urinary system, and reproductive system (Nayak P., Environmental Research Section TO (2002), 89: 101-115; Verstraeten SA et al., Arch Toxicol (2008) 82: 789-802; Flaten TP, Brain Research Bulletin (2001) 2: 187-196).

[0009] On the other hand, calcium phosphate (hereinafter CaP) is an inorganic substance that has been demonstrated to be useful as an immunological adjuvant. In US Patent No. 2,967,802, a mixture of erysipelas antigens and hydrated CaP gel is claimed. In research and subsequent patents, prophylactic vaccines against infections caused by several pathogenic and allergic microorganisms adsorbed to CaP were developed (US3,608,071, US4016252; US4350686; US20120121714; Courset, P. et al., Infect Immun (1986) 51(3):784-787).

[0010] As with hydroxides and aluminum phosphates, the immunological effect of CaP adjuvants is based on the formation of slowly released antigen depots, which allow for extended presentation to the immune system sufficient to generate a response to the inoculated antigen. US8,333,996 claims the development of immunogenic systems formed by adsorption of antigens such as: Bordetella pertusis, allergens, inactivated human immunodeficiency virus (HIV-2), keyhole limpet hemocyanin (KLH), diphtheria, tetanus toxoid, streptococci, attenuated bacteria or viruses, polio and vaccines against hepatitis A and B. They also include DNA and cytokine adsorption, such as granulocyte macrophage colony stimulating factor (GM-CSF).

[0011] The US6,355,271 patent claims a method for the preparation of CaP particles with a diameter of 300-4000 nm, which are based on an aqueous mixture of calcium chloride, sodium phosphate and sodium citrate, for adsorbing HSV-1, HSV-2, EBV virus, DNA, as well as ovovalbumin, a Mycobacterium tuberculosis antigen.

[0012] Recent studies have evaluated the use of CaP as a vaccine adjuvant and for the release of various pharmaceutical drugs. 10Research is relevant to evaluate the development of nanoparticles of (PO4)6(OH)2, hereafter HAp, preferably in the form of nanoparticles. This material is the main inorganic component of bone tissue, which ensures its biocompatibility. Furthermore, amorphous or poorly crystalline HAp is biodegradable in contact with biological media. The reduced particle size and the selected materials give it superior properties as an adjuvant compared to aluminum hydroxide. They generate a more balanced Th1 / Th2 response and produce less IgE-type antibodies (Qing, H. et al. Clinical and Diagnostic Laboratory Immunology (2000) 7(6):899-903; Jones, S. et al., Vaccine (2014) 32:4234-4242; Lin, Y. et al. Expert Review of Vaccines (2017), 16(9):895-906). The material is also biocompatible and biodegradable in amorphous form, reducing injection site side effects.

[0013] WO2005084637 patent claims a system comprising a CaP nanoparticle core, a biologically active macromolecule encapsulated in the core particle, and a surface modifier. The biologically active macromolecule can be a protein, a polypeptide, a polysaccharide, a nucleic acid, a polynucleotide, a lipid, or a carbohydrate.

[0014] On the other hand, WO2003051394 invention shows nanoparticulate HAp with different types of coatings in one formulation for use as an adjuvant, in which the nanoparticles are employed as carriers of native or recombinant antigens or other pharmacological agents applied to mucosal surfaces. Other studies claim the development and application of CaP in general, and HAp nanoparticles in particular, as immunological adjuvants or active substance carriers to be employed for the treatment of infectious or immune system diseases, including cancer (US5,443,832; US6,355,271; US6,767,550; US20020068090; US7,776,600; WO2017025359).

[0015] Previous reports have not described a vaccine composition that induces a specific immune response against EGF or other self-proteins, either independently of chemical binding of rhEGF molecules to a carrier protein constructed on the surface of CaP particles, or by conjugation of the two. Summary of the Invention

[0016] The novelty of the present invention is to provide a new vaccine composition for chronic treatment of cancer, which is based on biodegradable inorganic nanoparticles chemically linked to autoantigens. By binding nanometer or submicrometer sized CaP particles, especially HAp, with rhEGF or its peptides and another protein or carrier peptide capable of eliciting an immune response against EGF. These novel compositions are superior to existing compositions that elicit a response against EGF in that they have no side effects at the injection site and do not accumulate in the body. In addition, since they are dispensed into a single vial, no additional steps such as preparation of a mixture or emulsion are required at the time of use.

[0017] Brief description of the invention In one embodiment, the present invention relates to a vaccine composition for inducing an immune response against EGF. A system comprising rhEGF or a peptide thereof and a carrier protein or peptide linked to a core formed by biodegradable inorganic nanoparticles, which may be a salt, calcium oxide or hydroxide, iron, zinc, magnesium, zirconium, cerium, beryllium, silicon, or a mixture of two or more thereof, is activated. component Preferably, the inorganic core comprises CaP, more particularly of the HAp type, which is preferably amorphous or poorly crystalline and partially or completely coated with an organic ligand, in particular sodium citrate.

[0018] Further, the protein or peptide carrier is selected from the group comprising cholera toxin B subunit, tetanus toxoid, KLH, and P64k of Neisseria meningitidis.

[0019] In addition, the activity of the vaccine composition of the present invention component is on the surface of HAp nanoparticles bound by one of the following methods: - Chemical conjugates of rhEGF or its peptides with carrier proteins or peptides and covalently bonded to HAp nanoparticles (HAp-rhEGF-rP64k). - Covalent conjugation of rhEGF or its peptides with a carrier protein or peptide to HAp nanoparticles in an independent manner (HAp-PI). - Sequential covalent binding of rhEGF or its peptides to a carrier protein or peptide and to HAp nanoparticles. -Multiple conjugations between rhEGF or its peptides and carrier proteins or peptides on the surface of HAp (HAp-CM). - Encapsulation or physical adsorption of rhEGF and a carrier protein or peptide on the surface of HAp.

[0020] Another aspect of the present invention contemplates the combination of the vaccine compositions described herein with an adjuvant selected from the group including incomplete Freund's adjuvant, squalene-based adjuvants, adjuvants of synthetic origin, adjuvants of mineral origin, adjuvants of plant origin, adjuvants of animal origin, particulate protein adjuvants and liposomes.

[0021] In a further embodiment, an object of the present invention is the use of the vaccine composition described herein for the chronic treatment of cancer.

[0022] It is a further object of the present invention to provide a method of treatment of a subject in need thereof, comprising administering a therapeutically effective amount of the vaccine composition described herein, which method is particularly unique in that it maintains the induction of an immune response achieved with another vaccine composition against EGF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention The present invention involves the synthesis of biodegradable inorganic nanoparticles coated with organic ligands, their chemical activation, and the conjugation and assembly onto the surface of the particles of antigens that induce a specific immune response against EGF by covalent bonds or electrostatic interactions.

[0024] Obtaining and activating biodegradable inorganic nanoparticles The present invention involves forming biodegradable inorganic particles of nanometer, submicrometer or micrometer scale size, which are coated with an organic ligand, preferably sodium citrate, and obtained by precipitation, although this method should not be construed as limiting the scope of the invention.

[0025] The biodegradable particles have an inorganic core formed by a salt, oxide or hydroxide of calcium, iron, zinc, magnesium, zirconium, cerium, beryllium, silicon, or a mixture of two or more thereof, preferably formed by CaP, more preferably amorphous HAp, or amorphous HAp with one or more metals isomorphously substituted.

[0026] Inorganic particles are formed using sources of calcium and phosphate ions and organic ligands that endow them with surface charges and functional groups that allow for chemical bonding with biomolecules.

[0027] The particles used in the immunotherapeutic system according to the invention are characterized by having at least one of the following properties: They are formed by biodegradable inorganic particles which may have isotypic substitutions of metals or other ions. They are preferably obtained by wet synthesis, more preferably by precipitation, although this method should not be construed as limiting the scope of the invention. They have a micrometric, sub-micrometric or nanometric size, or a combination thereof. They may have a spherical, cylindrical or plate-like morphology or a combination thereof, but these morphologies should not be construed as limiting the scope of the invention. - They are preferably coated with organic ligands that endow them with surface charges and functional groups that allow conjugation with antigens.

[0028] To obtain the inorganic microparticles according to the invention, it is preferably necessary: - Maintain the molar ratio of cations to anions between 1:4. - Adding sodium citrate or other organic ligand having a similar effect on the reaction in an amount sufficient to provide a molar ratio of between 3:5 relative to the calcium ion content. - Adjusting the pH to 7-13 by adding alkali, preferably ammonium hydroxide or sodium hydroxide. - Carry out the reaction for 1 to 4 hours, preferably at room temperature (20±5°C). - The vacuum drying process is carried out at room temperature.

[0029] The inorganic particles, partially or completely coated with an organic ligand containing carboxylate groups, preferably sodium citrate, obtained under the conditions described above, are activated by adding a cross-linking reagent of the carbodiimide type or another with similar functionality. In this process, a mass ratio of reagent / particles of 1-6, preferably 2-5 mg / mg, is used. The suspension is kept under stirring at room temperature (20±5°C) for 1-4 hours, preferably 0.5-3 hours. The activated particles are then purified, preferably by a process of centrifugation or filtration.

[0030] The procedure described above results in surface-activated particles with a predominant size of ≦200 nm that have the ability to bind proteins with exposed functional groups. component This is superior to previous efforts developed from inorganic particles for the encapsulation or physical adsorption of proteins.

[0031] Achieving a nanoparticle system for the induction of anti-EGF responses The previously activated particles are mixed with a chemical conjugate from the recombinant proteins rhEGF and rP64k in a mass ratio of rhEGF-rP64k / HAp of 1-10, preferably 2-7 mg / mg. The previously described reaction couples the amine groups (NH2) of the conjugate with the carboxylate groups (COO - ) forms a covalent bond of amide type between the rhEGF-rP64k conjugate and the calcium groups remaining on the surface of HAp, which are negatively charged and expose carboxyl groups. The colloidal suspension is then dispersed, preferably with EDTA, and finally its pH is adjusted to a value between 6.7 and 7.3.

[0032] In another realization, the chemical conjugate of the recombinant protein rhEGF and rP64k is replaced by a conjugate of one or several peptides of rhEGF with rP64k or with another carrier protein.

[0033] The conjugate according to the present invention is attached to the surface of inorganic particles by the procedure defined above. This results in the first novel system of this type for the treatment of cancer, with a composition substantially different from the vaccine formulations described in US5,894,018 and US8,778,879. Inoculation of this system results in an immune response, including the production of anti-EGF antibodies, capable of capturing circulating EGF and blocking its binding to tumor surface receptors.

[0034] The systems obtained by covalent binding of antigens to inorganic particles are stable and can be stored for long periods, as required for parenteral formulations, a feature that makes them superior to immunotherapy or drug delivery systems previously developed starting from inorganic particles and using encapsulation or adsorption techniques of the active principle.

[0035] Conformation of conjugates on inorganic particles via covalent and electrostatic interactions for induction of anti-EGF responses The present invention includes other methods for the construction of systems that induce anti-EGF immune responses by covalent bonds and electrostatic interactions, which are described in detail below.

[0036] - Conjugation of rhEGF or peptides thereof and carrier proteins or peptides thereof to inorganic nanoparticles. The particles obtained by the methodology described above and previously activated with a carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), are mixed with a sterile phosphate buffer solution (PBS) of pH 7±0 containing rhEGF and rP64k in a rhEGF / rP64k molar ratio of 3 to 9, preferably 4 to 8. The mass ratios of HAp / rP64k and HAp / rhEGF used are 1 to 6, preferably 2 to 5 mg / mg. The suspension is then stirred at room temperature for 1 to 4 hours, more preferably 2 to 3 hours.

[0037] In a further realization, the recombinant protein rP64k is replaced by a carrier protein such as tetanus toxoid, KLH, etc. The invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0038] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0039] The above method results in a system in which rhEGF and rP64k protein are bound to the nanoparticles by chemical and electrostatic interactions without binding to each other. The inorganic particles constitute the structural elements for the linkage that ensure the immunogenicity of the system. Furthermore, it reduces the amount of rhEGF required to form the system by up to 40%, reducing the risks associated with the toxicity of glutaraldehyde used in the inventions of US5,894,018 and US8,778,879.

[0040] - Conjugation of rhEGF or its peptides with a carrier protein or its peptides in two reaction steps. The nanoparticles, obtained by the methodology described above and preactivated with a carbodiimide, preferably EDC, are mixed with a solution of PBS, pH 7±0.3, containing rP64k in a mass ratio of 1-6, preferably 2-5. The suspension is kept under stirring at room temperature for 1-4 hours, preferably 2-3 hours. The excess protein rP64k is then removed, preferably activated with EDC, then the excess EDC is removed and mixed with rhEGF in a molar ratio of 1-10, preferably 2-6, of rhEGF per rP64k.

[0041] In a further realization, tetanus toxoid, KLH or another carrier protein replaces the recombinant protein rP64k.The invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0042] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0043] This method produces a system in which a carrier protein is conjugated to biodegradable inorganic nanoparticles by chemical or electrostatic interactions, which are then conjugated with rhEGF. This method produces a rhEGF-rP64k conjugate formed from two layers of highly purified proteins, free of free rhEGF and rP64k. This conjugate is superior to the rhEGF-rP64k conjugates of the inventions of US5,894,018 and US8,778,879, and more than 40% of the obtained rhEGF is in free form, which is biologically irrelevant. It also reduces the risk associated with the toxicity of glutaraldehyde used in the two previous inventions.

[0044] - Multiple conjugation of rhEGF or its peptides and carrier proteins or its peptides to the surface of inorganic particles. The particles obtained by the methodology described above, preactivated with carbodiimide, preferably EDC, are mixed with a solution of rhEGF and rP64k in PBS, pH 7±0.3. Both proteins react with the activated particles and with the excess EDC that remains dissolved after activation, binding to each other and to the particle surface. To obtain this result, it is necessary to adopt a molar ratio of rhEGF / rP64k between 3 and 9, preferably between 4 and 8. The mass ratios of particles / rP64k and particles / rhEGF used are also between 1 and 6, preferably between 2 and 5. They are kept stirred at room temperature for 1 to 4 hours, more preferably 2 to 3 hours.

[0045] In a further realization, tetanus toxoid, KLH or another carrier protein replaces the rP64k recombinant protein.The invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0046] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0047] A complex matrix of rhEGF-rP64k conjugates is generated, and these proteins are linked to ligands that coat inorganic nanoparticles. As in previous realizations, this system also generates an immune response with anti-EGF antibodies. It also reduces the amount of rhEGF required to formulate the system by up to 40%, reducing the risks associated with the toxicity of glutaraldehyde used in the inventions of US 5,894,018 and US 8,778,879.

[0048] Construction of a system for inducing anti-EGF responses by encapsulation or adsorption onto inorganic particles. Although the chemically bonded system obtained by the above-mentioned procedure is more stable and suitable for vaccine formulation, encapsulation or adsorption of antigen is also a useful procedure. Therefore, the present invention provides a method for synthesizing immunogens in which inorganic core and rhEGF-rP64k conjugate or rhEGF and rP64k or its peptides are adsorbed inside or on the surface of particles.

[0049] - Encapsulation of rhEGF conjugates and carrier proteins or their peptides into inorganic particles. A source having calcium ions and phosphate ions is mixed in an aqueous solution, making sure that a suitable molar ratio of cations to anions of 1.4-3.5 is maintained. The rhEGF-P64k conjugate is added so that the mass ratio of rhEGF-rP64k / inorganic nanoparticles is 1-10, more preferably 2-7. Ammonium hydroxide or sodium hydroxide is then added until the pH is 8-10, and the reaction is continued at room temperature for 1-4 hours under stirring. Once the reaction is complete, it is preferably washed with purified water and separated by filtration. This procedure produces nanoparticles and microparticles containing rhEGF-rP64k conjugates in and on their surfaces, bound by interactions between the positively charged groups of the particles and the carboxylate groups of the conjugate, and between the negatively charged groups of the particles and the amine groups of the conjugate.

[0050] In a further realization, tetanus toxoid, KLH or another carrier protein replaces the recombinant rP64k protein.The invention also includes replacing the rP64k protein with one or several immunogenic peptides of natural or synthetic origin.

[0051] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0052] - Encapsulation of rhEGF or its peptides and a carrier protein or its peptides into inorganic particles. This procedure is similar to that described above, except that rhEGF and rP64k are added to the lysate in a mass ratio of 1-10, more preferably 2-7, instead of rhEGF-rP64k conjugate. During this process, a molar ratio of 1-10 rhEGF per rP64k, more preferably 2-5 rhEGF per rP64k, is maintained. Once the reaction is complete, it is preferably washed with purified water and separated by filtration. This procedure produces nanoparticles and microparticles that contain rhEGF-rP64k conjugates in their interior and on their surface, bound by electrostatic interactions between the positively charged groups of the particle and the carboxylate groups of the conjugate, and between the negatively charged groups of the particle and the amine groups of the conjugate.

[0053] In another realization, tetanus toxoid, KLH or another carrier protein replaces the recombinant protein rP64k.The present invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0054] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0055] - Adsorption of rhEGF conjugates and carrier proteins or peptides thereof onto inorganic particles. The aforementioned particles coated with sodium citrate are mixed with a PBS solution, pH 7±0.3, containing rhEGF-rP64k conjugate in a mass ratio of 1-10, more preferably 2-7. They are kept under stirring at room temperature for 1-4 hours, more preferably 2-3 hours. The system obtained from this procedure is dominated by nanoparticles with dimensions smaller than 200 nm. The rhEGF-rP64k conjugate is bound by electrostatic interactions between the positively charged groups on the nanoparticle surface and the carboxylate groups of the conjugate, and between the carboxylate groups of the citrate and the amine groups of the conjugate.

[0056] In other realizations, tetanus toxoid, KLH or other carrier proteins replace the recombinant protein rP64k.The present invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0057] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0058] - Adsorption of rhEGF or peptides thereof and carrier proteins or peptides thereof onto inorganic particles. A similar procedure to that described in the previous embodiment is carried out, with the only difference being that instead of rhEGF-rP64k conjugate, rhEGF and rP64k are added to the solution in a weight ratio of 1-10, more preferably 2-7. In this process, it is necessary to maintain a molar ratio of 1-10 rhEGF per rP64k, more preferably 2-5 rhEGF per rP64k. This procedure produces nanoparticles and microparticles with rhEGF and rP64k molecules on their surface, bound by interactions between positively charged groups of the particles and carboxylate groups of the protein, and between negatively charged groups of the particles and amine groups of the protein.

[0059] In a further realization, tetanus toxoid, KLH or another carrier protein replaces the recombinant protein rP64k.The invention also includes the replacement of the rP64k protein by one or several immunogenic peptides of natural or synthetic origin.

[0060] In a further realization, the rhEGF is replaced by one or several of the peptides.

[0061] The novel particulate systems mentioned above are formed on a biodegradable inorganic core to which is bound an autoantigen, preferably a chemical conjugate of the recombinant proteins rhEGF and rP64k, or said proteins or peptides thereof, individually. These systems are maintained by amide-type covalent bonds, encapsulation, or adsorption to the nanoparticle surface. They are dispersed with EDTA and, after adjusting the pH to 7±0.3, are the basis for parenterally administered cancer therapeutic vaccine formulations.

[0062] Pharmaceutical compositions and methods of treatment The pharmaceutical formulations obtained from the present invention are useful for treating cancers of epithelial tissue origin, such as: squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, head and neck and uterine cancer, glioblastoma, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer and penile cancer.

[0063] These can be used alone or in combination with adjuvants to enhance or complement the anti-EGF antibody response generated thereby.

[0064] In particular, the combination of biodegradable inorganic nanoparticle systems bound to rhEGF-rP64k protein with oleaginous adjuvants, such as incomplete Freund's adjuvants, squalene-based adjuvants, synthetic, mineral, vegetable or animal origin adjuvants, is an object of the present invention. Also included are particulate protein adjuvants and liposomes, or other adjuvants or systems capable of generating or enhancing humoral or cell-mediated immune responses, or combinations thereof. The above systems effectively maintain the previous induction of anti-EGF antibody immune responses generated in the formulations described in US8,778,879 patent, or in other systems that provide similar results.

[0065] The aforementioned combination is advantageous for the chronic treatment of cancers of epithelial origin, since treatment therewith avoids accumulation of foreign substances in the body at the injection site and therefore does not result in administration-related toxicity.

[0066] The anti-EGF system included in the present invention uses pharma- ceutical suitable excipients. These excipients include, but are not limited to, water for injection, sodium chloride, phosphorus and potassium salts, calcium chloride, sodium citrate, sodium hydroxide and EDTA. They can be administered to patients with epithelial tissue origin cancer as parenteral formulations at a protein concentration of 0.5-5 mg / mL, a dosage of 20-70 μL / kg or 20-70 μg / kg total protein or up to 5 mg total protein, more preferably in the range of 30-60 μg / kg. The dose of inorganic components defined in the present invention administered will be between 2-4.5 mg / kg, preferably 3-4 mg / kg, provided that it is within the approved dosage range for administration by intramuscular or subcutaneous routes in humans.

[0067] The present invention is further illustrated by the following examples and figures, which should not be construed as limiting the scope of the invention. [Brief description of the drawings]

[0068] [Figure 1] X-ray diffraction patterns: A) Amorphous HAp particles obtained in the present invention. B) Standard HAp pattern - JCPDS: PDF Ref. 09-0432. [Diagram 2] FTIR spectra: A) amorphous HAp particles obtained according to the invention. B) sodium citrate. [Diagram 3] Size of the amorphous HAp particles obtained according to the invention as determined by electron transmission microscopy. A) Image of the particles recorded at 25,000x magnification. B) Image of the particles recorded at 500,000x magnification. C) Size distribution of the particles. [Figure 4] 1 is a thermogram of amorphous HAp nanoparticles obtained according to the present invention. [Diagram 5]Size distribution of the covalently linked HAp-rhEGF-rP64k system determined by the method of dynamic light scattering (DLS).

[0069] [Figure 6] Characterization of the HAp-rhEGF-rP64k system by: A) SDS-PAGE: 1 - Molecular weight pattern. 2 - Positive control of rhEGF-rP64k conjugate produced according to the US8,778,879 invention. 3 - HAp-rhEGF-rP64k system. B) Western blot profile: 1 - Positive control of rhEGF-rP64k conjugate, 2 - HAp-rhEGF-rP64k system. [Figure 7] Anti-EGF antibody responses in C57BL / 6 mice covalently immunized with the HAp-rhEGF-rP64k system and the control group (Montanide-rhEGF-rP64k). [Figure 8] The ratio of antibody subclasses (IgG2b+IgG2c) / IgG1 in the sera of C57BL / 6 mice immunized with the HAp-rhEGF-rP64k system and the control group (Montanide-rhEGF-rP64k). [Figure 9] Photographs of the effects of injection sites of HAp-rhEGF-rP64k and Montanide-rhEGF-rP64k in BALB / c mice. A) Effects of Montanide-rhEGF-rP64k. B) Effects of HAp-rhEGF-rP64k. [Figure 10] Anti-EGF antibody responses in C57BL / 6 mice immunized with the HAp-rhEGF-rP64k system in combination with VSSP and with liposome-encapsulated rhEGF-rP64k.

[0070] [Figure 11] Ratio of antibody subclasses (IgG2b+IgG2c) / IgG1 in the sera of C57BL / 6 mice immunized with the HAp-rhEGF-rP64k system in combination with VSSP and with liposome-encapsulated rhEGF-rP64k. [Figure 12]Maintenance of anti-EGF antibody responses with the HAp-rhEGF-rP64k system, which had previously induced an immune response with one or two administrations of the Montanide-rhEGF-rP64k system. [Figure 13] Size distribution of HAp‐PI systems assembled on particles measured by DLS. [Figure 14] Characterization of the HAp-PI system by A) SDS-PAGE: 1-molecular weight pattern, 2-positive control of rP64k, 3-positive control of rhEGF, 4-HAp-PI system, and B) Western blot profile: 1-positive control of rP64k, 2-HAp-PI system. [Figure 15] Size distribution of HAp-CM systems measured by DLS.

[0071] [Figure 16] Characterization of the HAp-CM system by A) SDS-PAGE: 1 - molecular weight pattern, 2 - positive control of rhEGF-rP64k conjugate produced according to the US8,778,879 invention, 3 - HAp-CM system, and B) Western blot profile 1 - positive control of rhEGF-rP64k conjugate, 2 - HAp-CM system. [Figure 17] Anti-EGF antibody responses in C57BL / 6 mice immunized with HAp-rhEGF-rP64k, HAp-PI, and HAp-CM systems. [Figure 18] Ratio of antibody subclasses (IgG2b + IgG2c) / IgG1 in the sera of C57BL / 6 mice immunized with HAp-rhEGF-rP64k, HAp-PI and HAp-CM systems and Montanide-rhEGF-rP64k control. [Figure 19] Photographic images of tissue sections of muscle tissue extracted at the inoculation site and adjacent muscle tissue in C57BL / 6 mice, magnified 10x. * denotes fibroblastic repair tissue. Mice were administered: A-C: Montanide-rhEGF-rP64k, D: HAp-rhEGF-rP64k, E: HAp-CM, F: HAp-PI. [Figure 20]Number of lesions found in tissues at the injection site in C57BL / 6 mice immunized with HAp-rhEGF-rP64k, HAp-PI and HAp-CM systems, and Montanide-rhEGF-rP64k control. EXAMPLES

[0072] Example 1. Synthesis, characterization, and activation of HAp nanoparticles Nanoparticles of amorphous HAp coated with sodium citrate were obtained following a modified version of the procedure described by Koroleva M.,et al.,Russian Journal of Inorganic Chemistry(2016),61(6)674-680). Under controlled environmental conditions, a solution of 0.05 mol / L CaCl2 and sodium citrate was prepared in a molar ratio of sodium citrate / calcium of 4:1 (liquid A). Then, sterile solution B formed with 0.06 mol / L NaH2PO4 was added at a flow rate of 1 mL / min while maintaining a Ca / P molar ratio of 1.67. After that, the reaction solution was adjusted to pH 10 with a solution of sodium hydroxide and continued to stir at room temperature for 3 hours. After the reaction was completed, it was washed with purified water and filtered and separated using a 10 kDa Amicon polyethersulfone membrane. The obtained solid was dried under vacuum at room temperature. It was then sterilized with pure water vapor at 120 °C for 30 minutes and then dried under vacuum.

[0073] X-ray diffraction demonstrated the formation of amorphous HAp nanoparticles with a crystallinity of 9.3% and a crystallite size of 16.5 nm (Figure 1). The method used copper radiation (CuKα) in a tube operated at 45 kV and 40 mA, ranging from 10° to 90° with steps of 0.013° at 9 s intervals.

[0074] Figure 2 shows the Fourier transform infrared spectroscopy (FTIR) of nanoparticles (A) and sodium citrate (B) used as a control. In the nanoparticle spectrum, the peaks at 1090, 1030, 962, 604, 561, and 472 cm -1 The presence of a phosphate group corresponding to HAp was confirmed from the band observed at 3400 cm.-1 contains residual water and OH - A broad band due to the aryl group was observed in both spectra. -1 and 1413 cm -1 A band corresponding to the carboxyl group of citric acid was observed at 100 nm, confirming the presence of this ligand on the surface.

[0075] The nanoparticles exhibited a spherical morphology as determined by electron transmission microscopy (FIGS. 3A and 3B) with an average size of 62±13 nm (FIG. 3C). Thermography confirmed a mass loss of 6.2% at temperatures above 200 °C, which is associated with the presence of sodium citrate on the nanoparticle surface (Figure 4). The analysis was performed in the temperature range of 25 °C to 1000 °C, with a heating rate of 20 K / min and an argon flow rate of 60 mL / min.

[0076] Amorphous HAp nanoparticles were treated with a sterile dissolving solution of EDC at a mass ratio of EDC / HAp of 2:1 for 1 h under controlled environmental conditions, after which the suspension was centrifuged at 6708 g to remove excess EDC.

[0077] Example 2 Obtaining the HAp-rhEGF-rP64k system by covalent bonding between amorphous HAp nanoparticles and rhEGF-rP64k conjugate Nanoparticles of activated amorphous HAp obtained by the procedure described in Example 1 were mixed under controlled environmental conditions with a sterile solution of PBS, pH 7±0.3, containing the rhEGF-rP64k chemical conjugate obtained by the methodology described in the US8,778,879 invention, in a rhEGF-rP64k / HAp mass ratio of 1:2.5. The formed suspension was kept under shaking at room temperature for 2 hours at 140 cycles per minute.

[0078] The obtained HAp-rhEGF-rP64k system was dispersed in EDTA and adjusted to pH 7 ± 0.3 and protein concentration 1 ± 0.2 mg / mL with a sterile solution of PBS and sodium hydroxide.

[0079] DLS assay confirmed the presence of nanoparticles with a mean hydrodynamic diameter of 97.5 nm and a polydispersity index of 0.36. The resulting system was polydisperse, with particle sizes measured by intensity ranging from 9 to 366 nm (Figure 5).

[0080] The nanoparticle system obtained by the procedure described above was characterized by SDS-PAGE and Western blot (Figure 6). In electrophoresis, the rhEGF-rP64k conjugate had a band pattern similar to that of the positive control (i.e., a predominant band with a molecular weight of 66 kDa or more), indicating the presence of the conjugate in the system. In the Western blot assay, a band similar to that of the free rhEGF-rP64k conjugate control was observed, indicating the presence of rhEGF in the conjugate with a molecular weight of >200 kDa. This analysis demonstrated that the rhEGF-rP64k conjugate bound to the nanoparticles was able to maintain its structural and functional integrity, as confirmed by the fact that it was still recognized by anti-EGF antibodies.

[0081] Example 3 The HAp-rhEGF-rP64k system produces a humoral response to EGF without visible side effects at the injection site. C57BL / 6 (n=5) mice were immunized with 63 μg of protein contained in the system described in Example 2. A batch of product (Montanide-rhEGF-rP64k) obtained by the methodology described in the US8,778,879 invention was used as a positive control for the assay. An immunization protocol consisting of 4 doses was applied (days: 0, 14, 28, 42).

[0082] Total IgG antibody titers against EGF were measured by ELISA in both groups of mice 2 days before starting the immunization protocol and on days 35 and 56. Also, the ratio of EGF-specific (IgG2b+IgG2c) / IgG1 was measured in immune sera on day 56. Statistical analysis was performed using the Kruskal-Wallis mean comparison test, and different letters indicated statistically significant differences (p<0.05).

[0083] The HAp-rhEGF-rP64k system produced anti-EGF antibodies that could be detected in the immune serum at a dilution of 1 / 10000 on day 56 (Fig. 7). This result indicates that the binding of the rhEGF-rP64k conjugate to the particles does not affect the integrity of the conjugate and that HAp enhances the anti-EGF humoral response, albeit significantly lower than that of the control between groups. On the other hand, the ratio (IgG2b+IgG2c) / IgG1 showed no statistically significant difference between the responses obtained with the HAp-rhEGF-rP64k system and the control (Montanide-rhEGF-rP64k) (Fig. 8). In both systems, the predominant response was of the humoral type (Th2), making them very suitable for the depletion of EGF as part of the treatment of cancers of epithelial origin.

[0084] The effect of the HAp-rhEGF-rP64k system at the injection site was determined. For this purpose, five BALB / c mice were treated with the anti-EGF system (HaP-rhEGF-rP64k) described in Example 2 and five with the Montanide-rhEGF-rP64k control. The vaccination protocol was similar to that described previously. After the experiment was terminated, photographic images of the injection sites of the mice from the two treatment groups were taken. As can be seen in Figure 9A, the mice in the control group showed an accumulation of mineral oil at the injection site due to Montanide administration. On the other hand, in the mice treated with the HAp-rhEGF-rP64k system prepared according to the procedure described in Example 2 (Figure 9B), no damage was observed at the injection site. These results indicate that the novel system developed in this invention can significantly reduce the damage at the injection site and shows great potential for the treatment of cancers that require chronic administration, especially regular vaccination over several years.

[0085] Example 4 Combination of the HAp-rhEGF-rP64k system with a particulate adjuvant generates an anti-EGF IgG antibody response and induces a Th1-type response pattern. C57BL / 6 mice were used, divided into three groups of five. Animals were immunized as follows:

[0086] Group 1: 63 μg protein of the vaccine composition described in US Pat. No. 8,778,879 (Montanide-rhEGF-rhP64k) (positive control). Group 2: 63 μg protein of the HAp-rhEGF-rP64k system and 100 μg protein of the nanoparticle adjuvant VSSP. Group 3: 31.5 μg of protein of the HAp-rhEGF-rP64k system and 31.5 μg of rhEGF-rP64k conjugate encapsulated in liposomal vesicles (DRVs) obtained by the dehydration-rehydration methodology (Kirby and Gregoriadis, Biotechnology, (1984) 2:979-984).

[0087] Immunizations were performed on days 0, 14, 28, and 42. Blood samples were taken 2 days before the start of the protocol and on days 35 and 56, and total IgG antibody titers against EGF in the obtained serum were measured by ELISA. The ratio of EGF-specific (IgG2b+IgG2c) / IgG1 in serum was also determined on day 56. Statistical analysis was performed by the Kruskal-Wallis mean comparison test, and different letters indicate statistically significant differences (p<0.05).

[0088] The generation of anti-EGF antibodies was demonstrated in the three groups investigated. The combination of the HAp-rhEGF-rP64k system with VSSP and DRV liposomes produced antibodies that were detectable in immune sera up to a dilution of 1 / 10000 and were the same between the two extractions (Figure 10). The presence of a statistically significant difference of the combination with respect to the control was demonstrated.

[0089] When the EGF-specific ratio (IgG2b+IgG2c) / IgG1 was analyzed on day 56, both adjuvant combinations showed similar responses, and both were statistically superior compared to the control (Figure 11). These results demonstrated that the combination of HAp-based formulations with other particulate adjuvants enhanced the specific immune response against EGF inducing a superior Th1 response pattern that is most favorable for targeted cancer therapy.

[0090] Example 5 The HAp-rhEGF-rP64k system maintains the anti-EGF IgG antibody response previously induced with Montanide-rhEGF-rP64k. Three groups of C57BL / 6 mice (n=5) were immunized on days 0, 14, 28, 42, and 70 according to the following immunization schedule:

[0091] Group 1. (control) mice were immunized with 63 μg of protein contained in the system described in US Pat. No. 8,778,879 (Montanide-rhEGF-rhP64k). Group 2. Mice were immunized with 63 μg of protein from the Montanide-rhEGF-rhP64k system on day 0 and with the same amount of protein from the HAp-rhEGF-rhP64k system on the remaining immunizations. Group 3. Mice were immunized with 63 μg of protein from the Montanide-rhEGF-rhP64k system on days 0 and 14, and with the same amount of protein from the HAp-rhEGF-rP64k system on the remaining immunizations.

[0092] Preimmune serum was extracted 2 days before the first immunization, and immune serum was extracted on days 35, 56, and 84, and total IgG antibody titers against EGF were quantified by ELISA.

[0093] Anti-EGF antibody titers were detectable at the three time points studied, at 1 / 50000 dilution on day 35, and at 1 / 100000 dilution on days 56 and 84, without statistically significant differences among the three groups (Fig. 12). Inoculation with HAp-rhEGF-rP64k allowed the anti-EGF IgG antibody response induced by Montanide-rhEGF-rP64k to be maintained throughout the studied period. These results support the feasibility of using HAp instead of Montanide as an adjuvant for rhEGF-rP64k conjugates in the maintenance phase of anti-EGF immune responses, and its suitability in the chronic treatment of cancers of epithelial origin.

[0094] Example 6 Construction of HAp-PI system by covalent attachment of rhEGF and rP64k protein to the surface of amorphous HAp nanoparticles The sodium citrate pre-coated and EDC-activated HAp nanoparticles obtained according to the methodology described in Example 1 were mixed under controlled environmental conditions with a sterile PBS solution, pH 7 ± 0.3, containing rhEGF and rP64k in a molar ratio of 6 rhEGF per rP64k and a protein / HAp mass ratio of 1:2.5. The formed suspension was kept under shaking at 140 cycles per minute for 2 hours at room temperature. This procedure resulted in the rhEGF-HAp-rP64k system, characterized in that the proteins are bound to the HAp nanoparticles but not to each other. The system was dispersed with EDTA, the pH was adjusted to 7 ± 0.3 and the protein concentration to 1 ± 0.2 mg / mL.

[0095] Figure 13 shows the particle size profile measured by DLS. The average diameter was 111.2 nm and the polydispersity index was 0.347. The resulting system was similar to that produced in Example 2 in terms of size, polydispersity and DLS profile. This indicates that when rhEGF and rP64k were not bound to each other but bound to HAp nanoparticles, they had a dispersity similar to that of the rhEGF-rP64k conjugate.

[0096] As can be seen in Figure 14A, which shows the results of the SDS-PAGE assay, the lane corresponding to the HAp-PI system shows a main band at 66 kDa, characteristic of the rP64k protein, and another band at a height similar to that of the rhEGF control (6 kDa), indicating that both recombinant proteins are bound to the HAp nanoparticles and maintained their characteristic molecular weights even after reduction, as they are not bound to each other.

[0097] As shown in Figure 14B, there was a band at a height of approximately 66 kDa in lanes 1 and 2, confirming the presence of rP64k protein in the HAp-PI system and that the protein maintained its integrity when bound to HAp nanoparticles.

[0098] Example 7 Construction of HAp-CM system on the surface of amorphous HAp nanoparticles by multiple conjugation of rhEGF and rP64k protein Amorphous HAp nanoparticles obtained in Example 1 were treated with a sterile EDC solution for 1 h under controlled environmental conditions, keeping the mass ratio of EDC per HAp at 2.7. A sterile PBS solution (pH 7±0.3) containing rhEGF and rP64k in a molar ratio of 6 rhEGF per rP64k and a protein / HAp mass ratio of 1:2.5 was then added. The formed suspension was kept under shaking at 140 times per minute for 2 h at room temperature.

[0099] This procedure resulted in a system formed by HAp nanoparticles coated with both recombinant proteins bound to each other and to nanoparticles, which was dispersed with EDTA and adjusted to pH 7 ± 0.3, and then diluted with PBS solution until the protein concentration reached 1 ± 0.2 mg / mL.

[0100] The multiple conjugation described above resulted in the complex HAp-rhEGF-rP64k system with an average size of 90.2 nm and a polydispersity index of 0.337 as measured by DLS (FIG. 15).

[0101] Protein conjugation was confirmed by SDS-PAGE electrophoresis and Western blot characterization, yielding a product similar to that of the rhEGF-rP64k positive control recognizing anti-EGF antibody (Figure 16).

[0102] Example 8 A new system built on HAp nanoparticles generates anti-EGF IgG antibody responses and produces fewer epidermal lesions at the injection site Three groups of five C57BL / 6 mice were immunized with 63 μg of protein in the following systems:

[0103] Group 1: HAp-rhEGF-rhP64k system obtained according to the methodology described in Example 2. Group 2: HAp-PI systems obtained according to the methodology described in Example 6. Group 3: HAp-CM systems obtained according to the methodology described in Example 7.

[0104] Immunizations were performed on days 0, 14, 28, and 42. Blood samples were taken 2 days before the start of the protocol and on days 35 and 56. Total IgG antibody titers against EGF in the obtained serum were measured by ELISA. The ratio of EGF-specific (IgG2b+IgG2c) / IgG1 in serum was also determined on day 56. Statistical analysis was performed by the Kruskal-Wallis mean comparison test, and different letters indicate statistically significant differences (p<0.05).

[0105] The HAp-rhEGF-rP64k and HAp-CM systems produced detectable anti-EGF antibodies in immune sera up to 1 / 10000 dilution and the HAp-PI system up to 1 / 8000 dilution on days 35 and 56 (Figure 17). Confirming the results obtained by SDS-PAGE and Western blot (examples 6 and 7), it was demonstrated that all the systems constructed on HAp nanoparticles were immunogenic, even though rhEGF was not covalently bound to rP64k in the HAp-PI system. This system provides evidence that it is possible to create a system that induces an anti-EGF immune response without chemically binding rhEGF to a carrier protein.

[0106] Analysis of the (IgG2b IgG2c) / IgG1 ratio showed no statistically significant difference between the two responses. The predominant response was humoral type (Th2), which is appropriate for EGF depletion as part of the treatment of cancers of epithelial origin (Figure 18).

[0107] C57BL / 6 mice were sacrificed 120 days after the start of the experiment, and tissue specimens of the injection site were extracted for anatomopathological analysis, previously stained with hematoxylin-eosin. Tissue sections were fixed in neutral buffered formalin and processed by paraffin embedding. As can be seen from Figure 19A-C, which shows the results of the control group (Montanide-rhEGF-rP64k), there is a loss of normal structure of the muscle tissue at the inoculation site. The A zone is characterized by inflammatory infiltration and vascular dilation, with discontinuity between the adjacent muscle tissue. In Figure 19B and C, longitudinal (B) and transverse (C) fibers of the muscle tissue adjacent to the inoculation site with the infiltration of a fibroblastic repair tissue response can be observed. However, in Figure 19D-F, which shows the results of mice administered the novel system object of the present invention, normal structure can be observed at the inoculation site and adjacent muscle tissue. D: HAp-rhEGF-rP64k. E: HAp-CM. F: HAp-PI.

[0108] Mice from each of the three groups were evaluated for the presence or absence of the following lesions: -Discontinuity of the epidermis and lymphocytic infiltration of the dermis. - Inflammatory infiltrate. - Infiltration of muscle tissue. - Vacuolar degeneration of epithelial cells. -Dilation of blood vessels.

[0109] Each time a lesion was found, a point was added, and the values ​​corresponding to the mice in each group were summed and graphed.

[0110] This test demonstrated that the groups treated with the system developed in this invention showed a significantly reduced number of lesions, contrary to what was observed in the control group inoculated with the Montanide-rhEGF-rP64k system (Figure 20). This result confirms the visual observations of Example 3.

[0111] These results support the low toxicity of the HAp nanoparticle-based anti-EGF system and the side effects at the injection site due to the mineral oil contained in the Montanide-rhEGF-rP64k system.

Claims

1. A vaccine composition for chronic use to induce an immune response against epidermal growth factor (EGF), comprising as an active ingredient a system comprising recombinant human EGF (rhEGF) or a peptide thereof and a carrier protein bound to a core formed by inorganic nanoparticles of calcium phosphate.

2. 2. The vaccine composition according to claim 1, characterized in that the calcium phosphate is hydroxyapatite (HAp).

3. 2. A vaccine composition according to claim 1, characterized in that HAp is in amorphous form.

4. 2. The vaccine composition according to claim 1, characterized in that HAp has low crystallinity.

5. A vaccine composition according to any one of claims 1 to 4, characterized in that the hydroxyapatite is partially coated with an organic ligand.

6. 6. A vaccine composition according to claim 5, characterized in that the organic ligand is sodium citrate.

7. The carrier protein is - cholera toxin B subunit, - tetanus toxoid, -KLH and - P64k from Neisseria meningitidis 2. The vaccine composition according to claim 1, characterized in that it is selected from the group comprising:

8. A vaccine composition according to any one of claims 1 to 7, characterized in that the active ingredient is on the surface of the HAp nanoparticles.

9. Methods in which the active ingredient is: - chemical conjugates of rhEGF or peptides thereof with carrier proteins or peptides, and covalently bonded to HAp nanoparticles; - covalent binding of rhEGF or its peptides to carrier proteins or peptides and to HAp nanoparticles in an independent manner; - sequential covalent binding of rhEGF or a peptide thereof to a carrier protein or peptide and to HAp nanoparticles; - multiple conjugation of rhEGF or its peptides with carrier proteins or peptides on the surface of HAp, - encapsulation or physical binding of rhEGF to a carrier protein or peptide on the surface of HAp; 7. The vaccine composition according to claim 6, characterized in that the HAp nanoparticles are bound to the HAp nanoparticles by one of the following:

10. - incomplete Freund's adjuvant, - squalene-based adjuvants, - adjuvants of synthetic origin, - adjuvants of mineral origin, - adjuvants of plant origin, - adjuvants of animal origin, - particulate protein adjuvants, and -Liposomes The vaccine composition according to any one of claims 1 to 7, in combination with another adjuvant selected from the group comprising:

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