Composition for oral administration vaccine comprising fibroin-containing nanoparticles

Abstract

A composition for an orally administered vaccine, comprising nanoparticles containing an antigen and fibroin.

Description

Technical Field

[0001] The present invention relates to an orally administrable vaccine composition containing nanoparticles containing fibroin, and more specifically, to an orally administrable vaccine composition containing nanoparticles containing an antigen and fibroin, or an orally administrable vaccine composition containing nanoparticles containing an antigen, fibroin, and sericin. Background Art

[0002] In recent years, as a countermeasure to prevent and reduce the damage caused by chronic complex infections visible in the environment of mixed breeding of multiple livestock, a large amount of antibiotics have been used. Due to the abuse of antibiotics, the emergence of drug-resistant bacteria and the safety of food due to residues in food are a concern. As a substance to replace these antibiotics, the development of safe and useful animal vaccines has progressed. Further, in such vaccine development, an oral vaccine that can reduce the burden on animals and the risk to operators is desired compared to injectable vaccines.

[0003] In addition, although vaccination by injection can induce protective immunity and sensitization in the whole body system, it cannot defend through the respiratory / digestive mucosal tissues that are the portals of entry of pathogens. Especially in the case of infectious diseases, it can be said that the preventive effect of preventing infection is low.

[0004] Furthermore, it is very difficult to convert an injectable vaccine into an oral vaccine. The reason is that when administered orally, it must pass through the stomach, so it is often decomposed by the acidic environment in the stomach and digestive enzymes such as pepsin, and cannot accurately and effectively induce an antigen-specific immune response.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-97229

[0008] Non-Patent Documents

[0009] Non-Patent Document 1: Andreas S Lammel et al., Biomaterials, June 2010, Vol. 31, No. 16, pp. 4583 - 4591

[0010] Non-Patent Document 2: Zheng Zhao et al., Int J Mol Sci., March 4, 2015, Vol. 16, No. 3, pp. 4880 - 4903

[0011] Non-patent document 3: Fatemeh Rezaei et al., Biomater Sci., April 7, 2021, Vol. 9, No. 7, pp. 2679-2695 Summary of the invention

[0012] Problems to be solved by the invention

[0013] The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide a vaccine composition that can be orally administered to an animal to induce the production of antigen-specific antibodies.

[0014] Means for solving problems

[0015] In order to solve the above-mentioned problems, the present inventors conceived of using silk protein as a carrier for an orally administrable vaccine.

[0016] In addition, silk can be processed into powder, film, sponge, etc. by removing the outer sericin layer through scouring treatment, dissolving the obtained silk fibroin fibers into an aqueous solution. Furthermore, silk fibroin nanoparticles can be prepared from the silk fibroin aqueous solution by salting out, desolvation, etc. In addition, attempts have been made to use such nanoparticles as carriers for delivery systems of drugs, etc. (Non-patent Documents 1 to 3).

[0017] In addition, it has been reported that cocoons produced by genetically modified silkworms expressing antigenic proteins were physically crushed without scouring, and the resulting silk powder was injected subcutaneously into mice together with Freund's complete adjuvant, resulting in an increase in antibody titers in the blood (Patent Document 1).

[0018] However, the present inventors prepared silk powder according to the method described in Patent Document 1 and orally administered it to mice. The results showed that the production of antibodies against the silk component was induced predominantly, and it was difficult to efficiently induce the production of antibodies against the target antigen.

[0019] Therefore, the inventors have repeatedly conducted in-depth studies, and salted out a silk solution (a solution containing fibroin) in which scouring silk is dissolved or a silk solution (a solution containing sericin and fibroin) in which unscouring silk is dissolved with a mixed solution of antigen protein to produce silk nanoparticles containing antigen protein. It was also found that by orally administering the nanoparticles, it is possible to induce the production of antibodies specific to the above-mentioned antigen protein. In particular, in the above-mentioned antibody production, the induction of antigen-specific production of both IgA showing activation of mucosal immunity and IgG showing activation of systemic immunity was confirmed. That is, it is clear that the above-mentioned nanoparticles are carriers of excellent oral vaccines that avoid decomposition caused by gastric acid and digestive enzymes inside the stomach, deliver antigens to the intestines (small intestine, large intestine), thereby enabling mucosal immunity to work and inducing systemic immunity, thereby completing the present invention.

[0020] That is, the present invention provides the following solutions.

[0021] [1] An orally administrable vaccine composition comprising nanoparticles containing an antigen and fibroin.

[0022] [2] The orally administrable vaccine composition according to [1], wherein the nanoparticles further contain sericin.

[0023] [3] The orally administrable vaccine composition according to [1], wherein the nanoparticles are obtained by salting out from a mixed solution of a dissolution solution of refined silk and the antigen.

[0024] [4] The orally administrable vaccine composition according to [2], wherein the nanoparticles are obtained by salting out from a mixed solution of a dissolution solution of unrefined silk thread and the antigen.

[0025] Effects of the Invention

[0026] According to the present invention, by orally administering to an animal, it is possible to induce the production of antigen-specific antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A photograph showing the results of detecting the expression of the fusion protein (FibL-OVA) of fibroin light chain (FibL) and ovalbumin (OVA) in the cocoon produced by genetically engineered silkworms or the expression of free OVA (Ser-OVA) in the sericin layer by Western blotting using an anti-OVA antibody.

[0028] Figure 2 A graph showing the results of evaluating the antibody titer in the serum of mice after orally administering the disrupted product of the FibL-OVA expressing cocoon or Ser-OVA expressing cocoon suspended in physiological saline. In the graph, "unrefined cocoon silk", "refined silk", "sericin hope", and "OVA" on the horizontal axis show the results using test plates coated with an aqueous solution of unrefined cocoon silk, a dissolution solution of refined cocoon silk, an aqueous solution of sericin hope cocoon (containing only sericin and no fibroin), and an OVA solution, respectively, in ELISA. In the graph, "raw food" shows the results of the test area that was only orally administered physiological saline as a control.

[0029] Figure 3A A graph showing the process of preparing silk nanoparticles by mixing an antigen protein (OVA) in a dissolution solution of unrefined cocoon silk or a dissolution solution of refined cocoon silk.

[0030] Figure 3BPhotographs showing the results of observing the above-mentioned silk nanoparticles containing OVA using a scanning electron microscope (SEM) are presented. In the figures, "raw silk" shows the observation results of silk nanoparticles prepared only from the solution of unrefined raw silk, "raw silk + 10% OVA" shows the observation results of silk nanoparticles prepared by mixing OVA in this solution, "refined silk" shows the observation results of silk nanoparticles prepared only from the solution of refined raw silk, and "refined silk + 10% OVA" shows the observation results of silk nanoparticles prepared by mixing OVA in this solution.

[0031] Figure 4 Photographs showing the results of detecting OVA introduced into the above-mentioned silk nanoparticles by Western blotting using an anti-OVA antibody are presented.

[0032] Figure 5A A graph showing the results of analyzing the amount of IgG in the sera of mice orally administered the above-mentioned silk nanoparticles containing OVA by ELISA is presented. In the graph, "unrefined raw silk", "refined silk", "sericin desired", and "OVA" on the horizontal axis show the results obtained using assay plates coated with an aqueous solution of unrefined raw silk, a solution of refined raw silk, an aqueous solution of sericin-desired cocoons (containing only sericin protein without fibroin), and an OVA solution, respectively, in the ELISA. The legend "raw food" shows the results of the test area orally administered with physiological saline, "cocoon" shows the results of the test area orally administered with silk nanoparticles prepared only from the solution of unrefined raw silk, "cocoon + OVA" shows the results of the test area orally administered with silk nanoparticles prepared by mixing OVA in the solution of unrefined raw silk, "refined silk" shows the results of the test area orally administered with silk nanoparticles prepared only from the solution of refined raw silk, and "refined silk + OVA" shows the results of the test area orally administered with silk nanoparticles prepared by mixing OVA in the solution of refined raw silk.

[0033] Figure 5B A graph showing the results of analyzing the amount of IgA in the sera of mice orally administered the above-mentioned silk nanoparticles containing OVA by ELISA is presented. Regarding the descriptions in the graph, they are the same as those in Figure 5A those.

[0034] Figure 6Photographs showing the results of observing silk nanoparticles prepared using an aqueous solution in which various inorganic salts or organic salts are dissolved for salting out by SEM are presented. In the figures, "degummed silk" indicates that it is prepared from degummed cocoon silk, "cocoon" indicates that it is prepared from non-degummed cocoon silk, and "-OVA" or "+OVA" indicates that it is prepared without or with the antigen protein (OVA). The types of inorganic salts or organic salts used for salting out are shown on the left. The average particle size (diameter, μm) is shown below each photograph, and the value in parentheses indicates the standard deviation (SD). In addition, the average particle size here is the average value calculated from the maximum diameter of each particle present in one field of view of the SEM observation (the diameter of the smallest circle enclosing each particle). The number of particles detected was more than 50 except for the following two modes. (Mode 1) When there are only particles smaller than the measurement lower limit in one field of view of the SEM observation (in the figure, an example where the particle size becomes <0.34 μm), 5 to 20 particles were detected. (Mode 2) When there are 50 or fewer particles that can be measured in one field of view (sodium acetate: degummed silk - OVA, potassium acetate: degummed silk + OVA, etc.), all particles were detected as much as possible.

[0035] Figure 7 Photographs showing the results of detecting OVA introduced into silk nanoparticles prepared using an aqueous solution in which various inorganic salts or organic salts are dissolved by Western blotting using an anti-OVA antibody are presented. In the figures, "degummed silk + OVA" indicates silk nanoparticles prepared by including the antigen protein (OVA) in the dissolution solution of degummed cocoon silk, and "cocoon + OVA" indicates silk nanoparticles prepared from non-degummed cocoon silk and including OVA. The types of inorganic salts or organic salts used for salting out shown in each lane are as follows. In addition, the lane of "OVA" becomes the lane of the positive control. Lane No. 1: trisodium citrate, Lane No. 2: sodium sulfate, Lane No. 3: magnesium sulfate, Lane No. 4: magnesium acetate, Lane No. 5: sodium acetate, Lane No. 6: ammonium sulfate, Lane No. 7: potassium acetate, Lane No. 8: sodium tartrate, Lane No. 9: ammonium citrate. Detailed implementation mode

[0036] As shown in the following examples, the inventors have clarified that by orally administering nanoparticles containing an antigen prepared from a mixture of a dissolution solution of silk (a solution containing sericin and fibroin) and the antigen, it is possible to induce the production of antibodies specific to the antigen.

[0037] The inventors have also further clarified that by orally administering nanoparticles containing an antigen prepared from a mixture of a dissolution solution of degummed silk (a solution containing fibroin) and the antigen, it is possible to induce the production of antibodies specific to the antigen.

[0038] Therefore, the present invention relates to an orally administrable vaccine composition comprising nanoparticles containing an antigen and fibroin, and further relates to an orally administrable vaccine composition comprising the above nanoparticles further containing sericin.

[0039] <Regarding the nanoparticles of the present invention>

[0040] In the present invention, the "fibroin" contained in the nanoparticles refers to at least one protein selected from fibroin light chain, fibroin heavy chain, and Fibrohexamerin (P25) or a partial peptide of the protein. In addition, in the "fibroin" of the present invention, complexes of the above proteins are also included. As complexes, for example, naturally occurring states (e.g., a heterodimer formed by bonding of fibroin light chain and fibroin heavy chain through a disulfide bond, or a complex formed by aggregation of 6 molecules of the heterodimer and 1 molecule of Fibrohexamerin through non-covalent bonds) can be cited.

[0041] In the present invention, the "sericin" refers to at least one protein selected from sericin 1, sericin 2, sericin 3, and sericin 4 or a partial peptide of the protein. In addition, in the "sericin" of the present invention, complexes of the above proteins are also included. As complexes, for example, naturally occurring states (e.g., a sericin layer containing sericin 1, sericin 2, and sericin 3 or a fragment thereof) can be cited.

[0042] In the nanoparticles of the present invention, the above fibroin and sericin can form a complex and be contained. In addition, fibroin and sericin can be natural proteins derived from silk or recombinant proteins. As the "natural proteins", for example, proteins produced by lepidopteran insects (Bombyx mori, Antheraea pernyi, Antheraea yamamai, Antheraea militta, Antheraea assama, Philosamia cynthia ricini, bagworms, etc.), spiders (Araneae order), and arthropods belonging to Hymenoptera or Diptera can be cited. The "recombinant protein" refers to a protein prepared by genetic engineering methods. For example, it can be prepared by expressing a polynucleotide encoding the above protein in host cells such as Escherichia coli, yeast, insect cells, and animal cells, or in cell-free expression systems such as Escherichia coli extract, rabbit reticulocyte extract, and wheat germ extract.

[0043] In the present invention, "silk" refers to a fiber composed of fibroin fibers formed by the above-mentioned fibroin and a sericin protein layer that wraps the fibers. In addition, in the case of being derived from nature, it refers to fibers prepared from cocoons and the like produced by the above-mentioned arthropods. For example, cocoon layers, cocoon filaments, raw silk, and silk threads are included in the silk involved in the present invention. The so-called "scouring" refers to the treatment of removing sericin protein from silk. As long as this removal can be carried out, there is no particular limitation. Examples include alkali scouring, acid scouring, soap scouring, enzyme scouring, or a combination thereof (for example, soap / alkali scouring). However, from the viewpoint of cost and as a method with more practical achievements industrially, alkali scouring is preferred. The so-called alkali scouring refers to the treatment of boiling silk in the presence of an alkali. As the alkali used in this treatment, it is usually a weak alkali. Examples include sodium carbonate, sodium bicarbonate, and sodium silicate. Among them, from the viewpoints of cost and the solubility of the reagent, alkali scouring using sodium carbonate is more preferred.

[0044] In the present invention, in order to prepare nanoparticles, first, fibroin, and further sericin protein, scoured silk or unscoured silk are dissolved.

[0045] As the solvent used in such dissolution, there is no particular limitation, and water and / or organic solvents, etc. can be used. As organic solvents, for example, lower alcohols (ethanol, methanol, isopropyl alcohol, etc.), diols (propylene glycol, diethylene glycol, etc.) can be used, and aprotic solvents such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and N-methylmorpholine-N-oxide can be used. However, from the viewpoints of safety and environmental load, a mixed solvent of water and a lower alcohol is preferred, and a mixed solvent of water and ethanol is more preferred.

[0046] It is preferred to add inorganic salts to the solvent. As the inorganic salts, there is no particular limitation, and salts of alkali metals and alkaline earth metals, for example, can be used. Specifically, lithium bromide, potassium bromide, calcium bromide, lithium chloride, potassium chloride, calcium chloride, lithium thiocyanate, potassium thiocyanate, etc. can be cited. However, from the viewpoint of efficiently dissolving fibroin, sericin protein, and antigen protein while easily suppressing their decomposition, lithium bromide, lithium thiocyanate, and calcium chloride are preferred, and lithium bromide is more preferred.

[0047] In the present invention, as the concentration (final concentration) of the inorganic salts added to the solvent, there is no particular limitation, and it can be appropriately set to, for example, 3M or more, 4M or more, 5M or more, 6M or more, 7M or more, 8M or more, 9M or more, 10M or more. As the pH of the solvent, there is no particular limitation, but it is preferably pH 7 or more (for example, pH 8, pH 9).

[0048] The temperature during dissolution is not particularly limited, but is preferably 100 °C or lower, more preferably 55 °C or lower (such as 37 °C, room temperature (e.g., 25 °C), etc.). In addition, in order to remove the above-mentioned inorganic salts (desalination), the dissolution solution prepared by such an operation can be subjected to, for example, dialysis treatment. The dialysis solution used for such treatment is not particularly limited, and examples thereof include deionized water, RO water, or a liquid in which a buffer is added thereto (1 mM Tris-HCl (pH 9), PBS, etc.), or the above-mentioned solvent.

[0049] In the present invention, an antigen described below is mixed into the dissolution solution prepared by such an operation, and particle formation is carried out. As such a particle formation method, there is no particular limitation as long as the above-described nanoparticles can be prepared. Examples thereof include salting-out (salting-out method, see Lammel, A.S. et al., Biomaterials 2010, 31, 4583-4591, etc.), desolvation methods such as ethanol precipitation (coagulation method, see Kundu, J. et al., Int. J. Pharm. 2010, 388, 242-250, Zhang, Y.Q. et al., J. Nanopart. Res. 2007, 9, 885-900, Cao, Z. et al., Soft Matter 2007, 3, 910-915, Shi, P.J. et al., Int. J. Pharm. 2011, 410, 282-289, etc.), supercritical fluid technology (see Zhao, Z. et al., Ind. Eng. Chem. Res. 2013, 52, 3752-3761, etc.), electrospray method (see Gholami, A. et al., J. Nanopart. Res. 2011, 13, 2089-2098, Qu, J. et al., Mater. Sci. Eng. C: Mater. Biol. Appl. 2014, 44, 166-174, etc.), mechanical pulverization (see Rajkhowa, R. et al., Powder Technol. 2008, 185, 87-95, Rajkhowa, R. et al., Powder Technol. 2009, 191, 155-163, Kazemimostaghim, M. et al., Powder Technol. 2013, 241, 230-235, Kazemimostaghim, M. et al., Powder Technol. 2013, 249, 253-257, etc.), microemulsion method (see Myung, S.J. et al., Macromol. Res. 2008, 16, 604-608, etc.), electrical control method (see Huang, Y.L. et al., Chin. Sci. Bull. 2011, 56, 1013-1018, etc.), capillary microdroplet technology (see Gupta, V. et al., Int. J. Nanomed. 2009, 4, 115-122, etc.), PVA mixed film method (see Wang, X. et al., Biomaterials 2010, 31, 1025-1035, etc.).

[0050] Among these particle formation methods, from the viewpoints of the size, cost, and yield of the resulting nanoparticles, the ease of reducing the denaturation and decomposition of antigen proteins, and further the ease of avoiding the incorporation of other components into the nanoparticles, salting out and desolvation methods are preferred, and salting out is more preferred. In such salting out, as long as the antigen and fibroin can be precipitated by adding to the above-mentioned solution, there is no particular limitation, and it can be an inorganic salt or an organic salt. As the inorganic salt, for example, salts of alkali metals, alkaline earth metals, and ammonium salts can be used. Specifically, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, potassium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, sodium sulfate, ammonium sulfate, etc. can be cited. As the organic salt, trisodium citrate, triammonium citrate, magnesium acetate, sodium acetate, potassium acetate, and sodium tartrate can be cited. Among these salts, from the viewpoints of being difficult to be a component harmful to the inoculated animal and easily and efficiently recovering the nanoparticles by avoiding dilution, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium dihydrogen phosphate are preferred, and dipotassium hydrogen phosphate is more preferred.

[0051] In the present invention, the concentration (final concentration) of the salt added to the mixed solution of the antigen and the above-mentioned solution is not particularly limited as long as the antigen and fibroin can be salted out. For example, it can be 0.8 M or more, preferably 1 M or more, and more preferably 1.2 M or more. In addition, such salting out can be carried out by mixing a saturated aqueous solution of the salt into the antigen and the above-mentioned solution. The temperature during salting out is not particularly limited, but it is usually carried out at room temperature or below (for example, 25 °C, 4 °C).

[0052] In the present invention, the precipitate obtained by such an operation is washed with water, for example, and then broken, whereby nanoparticles can be prepared. As such a preparation method, there is no particular limitation, and for example, ultrasonic treatment and stirring can be cited.

[0053] The "nanoparticles" involved in the present invention are microparticles containing such silk proteins as carriers, and there is no limitation on their morphology. In addition, their particle size is in the nanometer size or micrometer size. The so-called "nanometer size or micrometer size" may include those with an average particle size of less than 5 μm, preferably less than 4 μm, more preferably less than 3 μm, further preferably less than 2 μm, more preferably less than 1 μm, further preferably 900 nm or less, more preferably 800 nm or less, further preferably 700 nm or less, more preferably 600 nm or less, further preferably 500 nm or less (for example, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less). In addition, the "average particle size" may include, for example, the average value of the particle sizes measured by observing the length through a scanning electron microscope (SEM). More specifically, it may include the average value calculated from the maximum diameter (including the diameter of the smallest circle of each particle) of each particle present in one field of view of the SEM observation. As the number of particles for detection, it may include, for example, 5 to 100 (for example, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 50 or more). In addition, it may be all present in one field of view.

[0054] Regarding the content of each protein in the nanoparticles involved in the present invention, when the nanoparticles contain an antigen and fibroin (for example, nanoparticles made from refined natural silk), the content of the antigen in the nanoparticles is preferably 0.05 to 30% by mass, more preferably 0.1 to 20% by mass, and further preferably 0.1 to 10% by mass relative to the content of fibroin in the nanoparticles. In addition, the mass ratio of fibroin to sericin in natural silk is about 3:1 (7:3 to 8:2). Furthermore, in the refining process, depending on the method and conditions, etc., sericin may not be completely removed from natural silk. Therefore, in such a case, in the nanoparticles involved in the present invention, sericin with a content of 0.1 to 20% by mass relative to the content of fibroin in the nanoparticles may be included as an impurity.

[0055] In addition, when the nanoparticles involved in the present invention contain an antigen, fibroin, and sericin (for example, nanoparticles made from unrefined natural silk), the content of the antigen in the nanoparticles is preferably 0.05 to 30% by mass, more preferably 0.1 to 20% by mass, and further preferably 0.1 to 10% by mass relative to the content of fibroin and sericin in the nanoparticles. In addition, the mass ratio of fibroin to sericin (the mass of fibroin in the nanoparticles: the mass of sericin in the nanoparticles) is preferably 60 to 90:10 to 40, more preferably 65 to 85:15 to 35.

[0056] The "antigen" contained in the nanoparticles of the present invention is not particularly limited as long as it can induce an immune response in animals, and examples thereof include viruses, bacteria, parasites, fungi, rickettsiae, chlamydiae, prions, cancer cells, or molecules derived therefrom (for example, proteins, nucleic acids, sugars, lipids).

[0057] Viruses, bacteria, parasites, fungi, rickettsiae, chlamydiae, etc. can be used as antigens (live vaccines) after attenuation, or can be used as antigens (inactivated vaccines) after inactivation. In addition, toxoids, capsids, surface proteins, etc. derived therefrom can be used as antigens.

[0058] The virus is not particularly limited as long as it can infect animals and cause diseases, and examples thereof include adenovirus, influenza virus, Ebola virus, Nipah virus, papillomavirus, human immunodeficiency virus, hepatitis virus (type A, B, C, D, E, F, G, etc.), measles virus, rubella virus, poliovirus, rotavirus, norovirus, sapovirus, enterovirus, rabies virus, yellow fever virus, varicella-zoster virus, mumps virus, cytomegalovirus, coronavirus, polyomavirus, herpes virus, Japanese encephalitis virus, dengue virus, Marburg virus, parvovirus, Lassa virus, hantavirus, Toscana virus, Dhori virus, Newcastle disease virus, togavirus, paramyxovirus, orthomyxovirus, poxvirus, reovirus, foot-and-mouth disease virus.

[0059] The bacterium is not particularly limited as long as it can infect animals and cause diseases, and examples thereof include Clostridium tetani, Streptococcus, Staphylococcus aureus, Enterococcus, Listeria, pathogenic Escherichia coli, Bordetella pertussis, Corynebacterium diphtheriae, Klebsiella pneumoniae, Proteus mirabilis, Neisseria meningitidis, Pseudomonas aeruginosa, Serratia marcescens, Neisseria gonorrhoeae, Enterobacter, Citrobacter, Mycoplasma, Bacillus, Mycobacterium tuberculosis, Vibrio cholerae, Yersinia pestis, Shigella, Bacillus anthracis, Treponema pallidum, Legionella pneumophila, Leptospira interrogans, Helicobacter pylori, Borrelia burgdorferi, Haemophilus influenzae.

[0060] The parasite is not particularly limited as long as it can parasitize animals and cause diseases, and examples thereof include Plasmodium, Toxoplasma gondii, Leishmania, Trypanosoma, Cryptosporidium, Taenia multiceps, Schistosoma, filaria, Ascaris lumbricoides.

[0061] The fungus is not particularly limited as long as it can infect animals and cause diseases, and examples thereof include Candida, Aspergillus, Cryptococcus, Histoplasma, Trichophyton, Pneumocystis, Coccidioides.

[0062] <Vaccine composition of the present invention, etc.>

[0063] The composition of the present invention is an orally administrable vaccine composition containing the above-mentioned silk particles. In such a composition, in addition to the silk particles, pharmaceutically acceptable additives may also be included. As such "pharmaceutically acceptable additives", for example, in the case where the composition of the present invention is in the form of a solid preparation, excipients, binders, lubricants, film coating agents, etc. can be cited. In the case where the composition of the present invention is in the form of a liquid preparation, isotonic agents, solubilizers, preservatives, stabilizers, suspending agents, emulsifying agents, etc. can be cited. In addition, the composition of the present invention may further contain an adjuvant. As adjuvants, for example, inorganic substances such as Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum gel adjuvant, microorganisms or substances derived from microorganisms (BCG, muramyl dipeptide, Bordetella pertussis, pertussis toxin, cholera toxin, etc.), surface active substances (saponins, deoxycholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil, etc.), alum can be cited.

[0064] In addition, in the present invention, as shown in the following examples, there is also provided a method of orally administering the nanoparticles or the orally administrable vaccine composition of the present invention to an animal to induce the production of antibodies specific to the above antigen in the animal. In addition, this method can also be said to be a method of preventing a disease (infectious disease, etc.) related to the above antigen or alleviating its symptoms.

[0065] As the "animal" that is the object of oral administration in the present invention, there is no particular limitation, and it can be a human or a non-human animal. As the "non-human animal", there is no particular limitation, and various domestic animals, poultry, pets, experimental animals, etc. can be cited. Specifically, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc. can be cited, but are not limited to these.

[0066] In addition, regarding the dosage of the nanoparticles or the orally administrable vaccine composition of the present invention, those skilled in the art can appropriately determine it according to the type of antigen, or the type, age, weight or health status of the administration object, etc. In addition, the number of administrations can be single, or can also be multiple, and this number can also be appropriately determined by those skilled in the art according to the type of the above antigen, etc.

[0067] Examples

[0068] Hereinafter, the present invention will be described more specifically based on examples and comparative examples, but the present invention is not limited to the following examples. In addition, the following examples were carried out using the following materials and methods.

[0069] (Materials and methods of the experiment)

[0070] 1. Oral administration test of mice using cocoons produced by recombinant silkworms as immunogens

[0071] According to the method described in Patent Document 1 (Japanese Patent Laid-Open No. 2005-97229), the cocoon silk of a genetically recombinant silkworm expressing an antigen protein was disrupted, and the resulting disrupted product was orally administered to evaluate whether antigen-specific antibody production was induced. Specifically, it was as follows.

[0072] 1-1. Disruption treatment of cocoons produced by recombinant silkworms expressing fibroin L chain-fused OVA protein (FibL-OVA) or cocoons produced by recombinant silkworms expressing free OVA (Ser-OVA) in the sericin protein layer

[0073] After cutting the FibL-OVA and Ser-OVA expressing cocoons into squares with a side length of 0.5 cm, cryogenic pulverization was carried out using a Multi-Beads Shocker MB3200(S) (Yasui Kikai Co., Ltd.) under liquid nitrogen conditions (repeating the cycle of 3 seconds of pulverization → 3 minutes of freezing with liquid nitrogen 15 times). Then, water (solvent) was added and wet cooling pulverization was carried out (repeating the cycle of 30 seconds of pulverization → 60 seconds of cooling (0°C) 40 times) to prepare silk powder with a particle size of several tens to several hundreds of μm.

[0074] 1-2. Oral administration test of FibL-OVA expressing silk powder or Ser-OVA expressing silk powder to mice

[0075] The concentration of the silk powder was calculated from the cocoon weight and suspended in physiological saline to be 50 mg / ml, and oral administration was carried out using an oral probe at a rate of 10 ml / kg (mouse body weight) per administration. An equal amount of physiological saline was orally administered as a control. After oral administration was carried out continuously for 3 days, oral administration was carried out 3 times every 1 week starting from the first administration, and whole blood was collected under isoflurane inhalation anesthesia on the day after the final administration. Three 6-week-old female ICR mice were used in each test control group.

[0076] 1-3. ELISA using the sera of mice orally administered with FibL-OVA expressing silk powder or Ser-OVA expressing silk powder

[0077] In a 96-well assay plate, 100 μl each of the undegummed cocoon solution or degummed silk solution of non-recombinant silkworm w1-pnd, the solution of sericin desired (consisting only of sericin protein) (each 0.25 mg / ml), or the OVA solution (0.625 mg / ml) was dispensed into each well and coated overnight at 4°C. Each well was blocked with Assay Diluent (Biolegend) (left to stand at room temperature for 60 minutes). After washing with PBS-T (PBS containing 0.05% Tween 20), a mouse serum diluted 1 / 200 with Assay Diluent was reacted (90 minutes at room temperature). After washing, it was reacted with an HRP-labeled anti-mouse immunoglobulin antibody (Dako) (60 minutes at room temperature). After washing, an ELISA POD substrate TMB solution (Nacalai Tesque) was added. After color development, 2N H2SO4 was added to stop the reaction, and the absorbance (450 nm) was measured (Bio-Rad, iMark Microplate Reader).

[0078] 2. Preparation of silk nanoparticles containing antigen protein using the salting-out method

[0079] 2-1. Preparation of cocoons and degummed silk

[0080] The cocoons used were those of w1-pnd. As the degummed silk, that obtained by subjecting the raw silk of "Gunma 200" to carbonate degumming (using a 0.02 M aqueous sodium carbonate solution, at 98°C, for 30 minutes) was used.

[0081] 2-2. Preparation of cocoon / degummed silk aqueous solution

[0082] After washing the cocoons or degummed silk with 70% ethanol and then with 0.1% SDS aqueous solution, they were thoroughly rinsed with RO water. For 2 g of degummed silk, 2 ml of 70% ethanol and 18 ml of 10 M LiBr, 100 mM Tris-HCl (pH 9.0) were added, and they were vigorously mixed at 37 °C to prepare an aqueous solution. For every 1 g of cocoons, 4 ml of 70% ethanol and 36 ml of 10 M LiBr, 100 mM Tris-HCl (pH 9.0) were added, and they were vigorously mixed at 37 °C to prepare an aqueous solution. The obtained aqueous solution of silk was sealed in a dialysis membrane (Aidis UC24-32-100) and dialyzed against RO water. Dialysis was continued while exchanging RO water until the conductivity of the external dialysis solution 4 hours after dialysis became 0.3 μS or less. The dialyzed aqueous solution of silk was recovered into a centrifuge tube, centrifuged at 12,000 rpm, 25 °C for 10 minutes (TOMY MX-307) to remove solid substances, and then diluted with RO water to prepare a 10 mg / ml aqueous solution.

[0083] 2-3. Preparation of silk nanoparticles (1)

[0084] To 50 ml of a 10 mg / ml aqueous solution of silk (cocoons or degummed silk), 50 ml of 1 mg / ml (w / w) OVA (FUJIFILM Wako 012-09885) or RO water was added and mixed, and then left standing at room temperature for 20 minutes to prepare a stock solution for nanoparticles.

[0085] In addition, the compositions of the 4 prepared stock solutions for nanoparticles are as described below.

[0086] (1) Cocoon dissolution solution (5 mg / ml)

[0087] (2) Cocoon + OVA cocoon dissolution solution (cocoon dissolution solution 5 mg / ml + OVA solution 0.5 mg / ml)

[0088] (3) Degummed silk dissolution solution (5 mg / ml)

[0089] (4) Degummed silk + OVA dissolution solution (degummed silk dissolution solution 5 mg / ml + OVA solution 0.5 mg / ml).

[0090] In a 500-ml centrifuge bottle, 360 ml of 1.25 M K2HPO4 was added, and 90 ml of the above-mentioned stock solution of nanoparticles was added thereto, followed by standing for 30 minutes. Then, the mixture was slowly inverted to mix, and nanoparticle formation was carried out by standing overnight at 4°C. After standing, centrifugation was performed (Himac RC-20, 4,000 rpm, 30 minutes, 25°C) to remove the supernatant, and the operation of adding 500 ml of RO water to the precipitate and centrifuging for washing was repeated twice. The obtained precipitate was suspended in a small amount of RO water and recovered in a 50-ml tube. After diluting to 50 ml with RO water, centrifugation was performed (TOMY MX-307; 5,000 rpm, room temperature, 10 minutes) to remove the supernatant, and the operation of washing with RO water was again repeated twice. 10 ml of RO water was added to the precipitate for suspension, and ultrasonic disruption (BRANSON SONIFIRE) was carried out to prepare a silk nanoparticle solution. Further, a portion of the silk nanoparticle solution was taken, 9 times the amount of 10 M LiBr and 100 mM Tris-HCl (pH 9.0) were added for dissolution, and the absorbance at 280 nm was measured. The protein concentration was measured based on the standard curve obtained from fibroin protein.

[0091] 2-4. Observation of Silk Nanoparticles Using Scanning Electron Microscope (SEM) (1)

[0092] The silk nanoparticles obtained in (2-3) were diluted 10-fold with RO water, and 1 μl was dropped onto a silicon wafer to prepare a sample for observation. Observation was carried out using a bench microscope (HITACHI TM2000Plus) under the conditions of 5 kV and 2,500 times magnification.

[0093] 2-5. Confirmation of Introduction of Antigen Protein into Silk Nanoparticles (1)

[0094] To the "silk fibroin nanoparticles containing OVA" or "degummed silk nanoparticles containing OVA" prepared in (2-3), 10 M LiBr and 100 mM Tris-HCl (pH 9.0) were added for dissolution, and the concentration was adjusted to 2 mg / ml (in 2 M LiBr, 20 mM Tri-HCl (pH 9.0)) aqueous silk solution. Further, serial dilutions were performed with SDS sample buffer, and samples were loaded into each well to be 5, 2.5, 1, 0.5, 0.25 μg. In addition, the nanoparticle suspension was stored at 4 °C for about 1 week, a part of its supernatant was recovered, diluted 1 / 20 with SDS sample buffer, and 5 μl of it was subjected to electrophoresis together. After SDS-PAGE, it was transferred to a PVDF membrane, reacted with an anti-OVA antibody (Abcam) (room temperature, 60 minutes), and then reacted with an alkaline phosphatase-labeled anti-rabbit antibody (Dako) (room temperature, 60 minutes). A BCIP-NBT solution (Nacalai Tesque) was added to cause a color development reaction.

[0095] 2-6. Preparation of Silk Nanoparticles (2)

[0096] In addition, instead of the above 1.25 M K2HPO4, various salting-out solvents prepared at a concentration of 1.25 M were used, and attempts were made to prepare silk nanoparticles on a smaller scale. Specifically, 1.5 ml of each of the various salting-out solvents (sodium citrate, sodium sulfate, magnesium sulfate, magnesium acetate, sodium acetate, ammonium sulfate, potassium acetate, sodium tartrate, or ammonium citrate) prepared at a concentration of 1.25 M in advance were added to a 2-ml tube, and 0.3 ml of the above stock solution for nanoparticles was overlaid thereon, and left standing for 30 minutes. Then, it was slowly inverted and mixed to mix, and left standing overnight at 4 °C for nanoparticle formation. After standing, centrifugation was performed (TOMY MX-307, 5,000 rpm, 15 °C, 20 minutes) to remove the supernatant, 1.5 ml of RO water was added to the precipitate and inverted and mixed, centrifugation was performed (TOMY MX-307, 15,000 rpm, 15 °C, 20 minutes) to remove the supernatant, 250 μl of RO water was added to the precipitate, and ultrasonic disruption (BRANSON SONIFIRE) was performed to prepare a silk nanoparticle solution.

[0097] 2-7. Observation of Silk Nanoparticles by SEM (2)

[0098] 1 μl of the silk nanoparticles obtained in (2-6) was dropped onto a silicon wafer and dried to prepare a specimen for observation. Observation was performed using a bench-top microscope (HITACHI TM4000Plus) under the conditions of 5 kV and 2500 times magnification.

[0099] Confirmation of introduction of antigen protein into silk nanoparticles (2)

[0100] Take 150 μl of the silk nanoparticle solution prepared in (2-6), centrifuge (15,000 rpm, 15 °C, 10 minutes), discard 140 μl of the supernatant (10 μl remains), add 40 μl of 10 M LiBr and 100 mM Tris-HCl (pH 9.0), and dissolve completely. Measure A280 to calculate the protein concentration and adjust it to 1.5 mg / ml in 2 M LiBr, 20 mM Tris-HCl (pH 9.0).

[0101] Add an equal volume of 2X SDS sample buffer and boil. Load the samples into each well to make 3 μg. After SDS-PAGE, transfer to a PVDF membrane. After reacting with anti-OVA antibody (Abcam) (room temperature, 60 minutes), react it with horseradish peroxidase (HRP)-labeled anti-rabbit antibody (Dako) (room temperature, 60 minutes). Add the chemiluminescence analysis reagent Chemi-Lumi One L (Nacalai Tesque) to cause chemiluminescence and develop using Hyperfilm ECL (Cytiva).

[0102] 3. Oral administration test of silk nanoparticles containing OVA to mice

[0103] 3-1. The 4 types of silk nanoparticles (about 35 mg / ml) prepared in the above (2-3) were orally administered at a rate of 10 ml / kg (mouse body weight) per administration using an oral probe. An equal volume of physiological saline was orally administered as a control. After oral administration for 3 consecutive days, oral administration was performed 3 times every 1 week starting from the first administration. Whole blood was collected under isoflurane inhalation anesthesia on the day after the final administration. Three 6-week-old female ICR mice were used in each experimental control group.

[0104] 3-2. ELISA using mouse serum orally administered with silk nanoparticles containing OVA

[0105] In a 96-well assay plate, 100 μl each of unrefined silk filaments, refined silk, sericin solution for desired dissolution (each at 0.25 mg / ml), or OVA solution (0.625 mg / ml) was dispensed into each well and coated overnight at 4°C. Each well was blocked with Assay Diluent (Biolegend) (left standing at room temperature for 60 minutes). After washing with PBST (PBS containing 0.05% Tween 20), mouse serum diluted 1 / 100 with Assay Diluent was reacted (90 minutes at room temperature). After washing, it was reacted with HRP-labeled anti-mouse immunoglobulin antibody (Dako) or HRP-labeled anti-mouse IgA antibody (Abcam) (60 minutes at room temperature). After washing, ELISA POD substrate TMB solution (Nacalai Tesque) was added. After color development, 2N H2SO4 was added to stop the reaction, and the absorbance (450 nm) was measured (Bio-Rad, iMark Microplate Reader).

[0106] The results obtained using the above materials and methods are shown below.

[0107] (Results)

[0108] (Comparative Example 1) Oral administration test using cocoons produced by recombinant silkworms as immunogens

[0109] Cocoons produced by recombinant silkworms expressing fibroin light chain fusion OVA protein (FibL-OVA) or cocoons produced by recombinant silkworms expressing free OVA (Ser-OVA) in the sericin protein layer were completely dissolved with 9M lithium bromide. The silk filament lysate was subjected to SDS-PAGE and then Western blotting using an anti-OVA antibody (Abcam) (primary antibody: anti-OVA antibody, secondary antibody: alkaline phosphatase-labeled anti-rabbit polyclonal antibody). As a result, in the silk filaments produced by recombinant silkworms, the expression of each of the FibL-OVA fusion protein and free OVA (Ser-OVA) in the sericin protein layer could be confirmed ( Figure 1 ).

[0110] Then, silk microparticles obtained by crushing Fib-OVA or Ser-OVA-expressing cocoons were orally administered to mice. As a result, in the sera of these mice, meaningful induction of antibody production against OVA could not be confirmed. On the other hand, antibody production was strongly induced against silk components, especially sericin protein ( Figure 2 ).

[0111] In summary, the results of orally administering the crushed product of the cocoon produced by a genetically engineered silkworm expressing an antigen protein, prepared as described in Japanese Patent Application Laid-Open No. 2005-97229, suggest that antibody production against the silk component is predominantly induced, and it is difficult to efficiently induce antibody production against the target antigen.

[0112] (Example 1) Oral administration test of mice using silk nanoparticles containing OVA as an immunogen

[0113] A silk nanoparticle was prepared by mixing a mixture of 10% of the amount of OVA as the silk protein added to the cocoon dissolution solution or the degummed silk dissolution solution with 1.25 M K2HPO4 ( Figure 3A ). The obtained silk nanoparticles were observed by scanning electron microscopy (SEM). As a result, the silk nanoparticles using degummed silk had the largest diameter, which was 0.5 - 4 μm, and there were many particles with an overall diameter of about 2 μm. On the other hand, for the nanoparticles added with 10% OVA, although the particle size distribution was almost the same, a large number of slightly smaller particles with a diameter of 0.5 - 1.0 μm were confirmed. When the cocoon dissolution solution or the cocoon dissolution solution containing 10% OVA was used as the raw material, the particles became even smaller, and particles with a diameter of 0.2 - 0.3 μm accounted for the majority ( Figure 3B ).

[0114] In addition, in order to confirm that OVA was introduced into the prepared silk nanoparticles, the prepared silk nanoparticles were dissolved again with lithium bromide, and after SDS-PAGE of the dissolution solution, Western blotting was performed using an anti-OVA antibody to confirm that OVA was indeed introduced into the silk nanoparticles ( Figure 4 ).

[0115] Furthermore, it was confirmed that OVA was present in the supernatant obtained by storing the suspension of silk nanoparticles prepared using the cocoon dissolution solution containing 10% OVA or the degummed silk dissolution solution containing 10% OVA at 4°C for about 1 week. Therefore, it was confirmed that once OVA was introduced into the silk nanoparticles, it was gradually released into the solution in which the nanoparticles were suspended ( Figure 4 ).

[0116] Then, using the sera of mice orally administered with silk nanoparticles prepared using the cocoon dissolution solution, the cocoon + OVA cocoon dissolution solution, the degummed silk dissolution solution, or the degummed silk + OVA dissolution solution as raw materials, it was evaluated whether antibody production against the silk component and OVA was induced.

[0117] As a result, in the sera of mice orally administered with nanoparticles composed only of cocoons, the production of antibodies (IgG and IgA) specific to sericin, a silk fibroin component, was confirmed. In addition, although rarely, antibodies were also induced against fibroin because they reacted with the degummed silk lysate from which sericin had been removed ( Figure 5A and 5B ).

[0118] On the other hand, it was clarified that in mice orally administered with nanoparticles prepared from cocoons + OVA cocoon lysate, although the production of antibodies (IgG and IgA) specific to sericin was also induced, the production of antibodies (IgG and IgA) specific to OVA was further induced ( Figure 5A and 5B ).

[0119] In addition, in mice orally administered with nanoparticles composed only of degummed silk or nanoparticles prepared from degummed silk + OVA lysate, neither the induction of antibody production against the removed sericin nor the induction of antibody production against fibroin was confirmed ( Figure 5A and 5B ).

[0120] Furthermore, in mice orally administered with nanoparticles prepared from degummed silk + OVA lysate, the production of antibodies (IgG and IgA) specific to OVA was significantly induced ( Figure 5A and 5B ).

[0121] (Example 2) Formation of silk nanoparticles containing OVA using various salting-out solvents

[0122] As shown in (2-6) above, a mixture of 10% of the amount of OVA added to the cocoon lysate or degummed silk lysate was mixed with various salting-out solvents prepared to 1.25 M to produce silk nanoparticles. The resulting silk nanoparticles were observed by SEM, and as a result, it was confirmed that particles with an average diameter of about 1-4 μm were formed when only the degummed silk lysate (degummed silk-OVA) was used. On the other hand, for the case where 10% OVA was added to the degummed silk lysate (degummed silk + OVA), it was confirmed that particles with a slightly smaller diameter of 0.8-3 μm were formed under each condition. When using only the cocoon lysate (cocoon-OVA) or a liquid with 10% OVA added to the cocoon lysate (cocoon + OVA) as the raw material, it was confirmed that the particles became further smaller, forming particles with an average diameter of less than 0.3 μm to about 0.5 μm ( Figure 6 ).

[0123] In addition, in order to confirm that OVA was introduced into the silk nanoparticles produced by such an operation, the produced silk nanoparticles were dissolved again with lithium bromide, and after SDS-PAGE of the dissolved solution, Western blotting was performed using an anti-OVA antibody. As a result, it was confirmed that OVA was indeed introduced into each of the silk nanoparticles produced using different salting-out solvents( Figure 7 ).

[0124] In summary, it was clarified that oral administration of silk nanoparticles containing an antigen, which were produced from a silk solution (a solution containing fibroin) in which degummed silk was dissolved or a silk solution (a solution containing sericin and fibroin) in which non-degummed cocoon silk was dissolved and a mixture of an antigen protein, could induce the production of antibodies specific to the antigen.

[0125] Industrial Applicability

[0126] As described above, according to the present invention, oral administration of nanoparticles containing an antigen and fibroin can induce the production of antigen-specific antibodies. In particular, in the above antibody production, induction of the production of antigen specificity of both IgA showing activation of mucosal immunity and IgG showing activation of systemic immunity was confirmed. That is, the above nanoparticles avoid decomposition by gastric acid and digestive enzymes in the stomach and transfer the antigen to the intestine (small intestine, large intestine), thereby activating mucosal immunity and also inducing systemic immunity. Therefore, the present invention is useful as an orally administered vaccine composition.

Claims

1. An orally administered vaccine composition comprising nanoparticles containing an antigen and fibroin.

2. The orally administered vaccine composition according to claim 1, wherein the nanoparticles further contain sericin.

3. The orally administered vaccine composition according to claim 1, wherein the nanoparticles are obtained by salting out from a mixed solution of a dissolution solution of refined raw silk and the antigen.

4. The orally administered vaccine composition according to claim 2, wherein the nanoparticles are obtained by salting out from a mixed solution of a dissolution solution of unrefined raw silk thread and the antigen.

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