US20160083437A1 - Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging - Google Patents

Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging Download PDF

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US20160083437A1
US20160083437A1 US14/764,466 US201314764466A US2016083437A1 US 20160083437 A1 US20160083437 A1 US 20160083437A1 US 201314764466 A US201314764466 A US 201314764466A US 2016083437 A1 US2016083437 A1 US 2016083437A1
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composition
recombinant protein
present
preventing
immunization
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Kyung A. Cho
Jae Sung Lim
Joon Haeng Rhee
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Industry Foundation of Chonnam National University
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Industry Foundation of Chonnam National University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/107—Vibrio
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention was made with the support of the National Research Foundation of Korea, under Project No. 2012-0002472, which was conducted in the program titled “General Researcher Support Project/Female Engineer Support Project” in the project named “Research for the Mechanism of Aging Dependent Innate immune dysfunction” by the Chonnam National University under the management of the National Research Foundation of Korea, from May 1, 2010 to Apr. 30, 2013.
  • the present invention was made with the support of the Ministry of Knowledge Economy, Republic of Korea, under Project No. RTI-0501-01, which was conducted in the program titled “New Vaccines and Immune Disease Therapeutic Agents Development Project” in the project named “Development of Anti-aging Vaccine Adjuvant” by the Chonnam National University under the management of the Ministry of Knowledge Economy, Republic of Korea, from Jul. 1, 2011 to Jun. 30, 2012.
  • the present invention was made with the support of the Ministry of Education, Science, and Technology of Republic of Korea, under Project No. 2011-0030034, which was conducted in the program titled “Overseas Excellent Research Institutions Inducement Project” in the project named “Age-Related Cellular Function Regulation” by the Chonnam National University Hwasun Hospital under the management of the Ministry of Education, Science, and Technology of Republic of Korea, from Jul. 1, 2011 to Jun. 30, 2012.
  • the present invention relates to a composition for preventing, improving, or treating aging, the composition comprising a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella and an agonist of a toll-like receptor 5 (TLR-5), fused with a pathogenic antigen, and to a method for preventing, improving, or treating aging.
  • a recombinant protein of flagellin which is the constituent of Vibrio vulnificus flagella and an agonist of a toll-like receptor 5 (TLR-5), fused with a pathogenic antigen
  • the aging process causes a wide variety of changes.
  • Various changes appear, including reduced functions of respective main tissues, food intake and digestive disorders, reduced brain functions including defective memory, and reduced cardiovascular functions, as well as various appearance changes, such as skin wrinkles, hair decoloration, spine curving, and the change in motion.
  • these changes induce the reduction of functions and diseases of the respective tissues, and therefore, it is very important to understand causes of the reduction of external and internal functions due to aging and develop techniques of regulating the functions.
  • one of the large changes in functions due to aging is the reduction in immune function. This is called immunosenescence.
  • pathogens invade a host
  • the host defection action is made by two immune systems, an innate immune system and an adaptive immune system.
  • the innate immune system is activated immediately after infection, to promptly regulate infecting pathogens, and takes charge of the initial infection until the adaptive immune system is activated.
  • a receptor recognizing “pathogen associated molecular patterns (PAMPs) existing in the pathogens is called “pattern recognition receptors (PRRs), and these receptors are called toll-like receptors in a mammal.
  • PAMPs pathogen associated molecular patterns
  • TLRs TLRs
  • PRRs like TLRs exist on the cell surface or in the protoplasm, and have been known to regulate the innate immune response by various stimuli of PAMPs, and further regulate the adaptive immune response. Therefore, the agonists of TLRs may be target materials suitable for the development of various immunomodulators and vaccine adjuvants.
  • the present inventors have endeavored to develop a material capable of preventing, improving, or treating dysfunction due to aging. As a result, the present inventors have confirmed that the expressions of most toll-like receptors are reduced but toll-like receptor-5 is well expressed to keep functions thereof in aging immune cells, and confirmed that, when aged mice are immunized with a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen of a pathogen, the immunity of the aged mice is activated and external and internal functions of the aged mice are improved, and thus have completed the present invention.
  • flagellin which is the constituent of Vibrio vulnificus flagella
  • an object of the present invention is to provide a composition for preventing, improving, or treating aging, the composition comprising, as an active ingredient, a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen.
  • Another object of the present invention is to provide a composition for preventing, improving, or treating hair-related disease, the composition comprising the recombinant protein as an active ingredient.
  • Still another object of the present invention is to provide a composition for preventing, improving, or treating eye-related disease, the composition comprising the recombinant protein as an active ingredient.
  • Still another object of the present invention is to provide a composition for preventing, improving, or treating bowel disease, the composition comprising the recombinant protein as an active ingredient.
  • Still another object of the present invention is to provide a composition for preventing, improving, or treating bone disease, the composition comprising the recombinant protein as an active ingredient.
  • Another object of the present invention is to provide a method for preventing, improving, or treating aging.
  • the present invention provides a composition for preventing, improving, or treating aging, the composition comprising, as an active ingredient, a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen.
  • the present inventors have endeavored to develop a material capable of preventing, improving, or treating dysfunction due to aging. As a result, the present inventors have confirmed that the expressions of most toll-like receptors are reduced but toll-like receptor-5 is well expressed to keep functions thereof in aging immune cells, and confirmed that, when aged mice are immunized with a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen, the immunity of the aged mice is activated and external and internal functions of the aged mice are improved.
  • flagellin which is the constituent of Vibrio vulnificus flagella
  • the largest feature of the present invention is to use a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen.
  • flagellin refers to a unit molecule constituting flagella, which are the cilia to determine the mobility of bacteria.
  • the flagellin of the present invention includes all flagellins that act as a TLR-5 activator of Vibrio vulnificus .
  • the flagellin of the present invention is FlaB, which is the flagellin of Vibrio vulnificus.
  • the pathogenic protein antigen usable herein may include an ⁇ -helix domain of surface protein A (PspA) and pneumococcal surface protein A (PsaA) of Streptococcus pneumonia ; subunit hemagglutinin (HA) and neuraminidase (NA) of influenza virus; and spike (S) protein of severe acute respiratory syndrome virus (SARS virus), but are not limited thereto.
  • the pathogenic protein antigen is surface protein A (PspA) of Streptococcus pneumonia.
  • the recombinant protein of the present invention is FlaB-PspA protein having an amino acid sequence of SEQ ID NO: 1.
  • aging refers to a functional, structural, and biochemical procedure that continuously occurs in a subject from birth until death. The aging occurs in the overall cells and body tissues constituting the subject, and indicates the decrease in the metabolic rate, the increase in diseases, and the deterioration in adaptability, ultimately leading to death in cells and the whole body.
  • examples of aging include external changes, such as the increase in skin wrinkles, the reduction of hair gloss, hair decoloration, hair loss, the thickness reduction of the dermal layer in which hair follicles are present, the reduction in the number of follicles, spine curving, and the reduction in exercising; internal changes, such as the reduction in immune functions and the reduction in functions of main tissues; and the consequent occurrence of diseases of respective tissues, but are not limited thereto.
  • the recombinant protein of the present invention enhances immunity.
  • enhancing immunity refers to keeping the immune response or activity of the in vivo immune system at a level of a non-aged control group or enhancing the same to a level of an aged control group.
  • the recombinant protein of the present invention prevents the reduction of functionality of immune-related organs due to aging or enhances the functionality compared with the aged control group, by increasing the production of secretory globulin A (secretory lgA, slgA) antibody, increasing the frequency of hematopoietic stem cells which are essentially associated with T cell differentiation, preventing thymic involution, or preventing the hypertrophy of mesenteric lymph nodes (MLNs) or spleens.
  • secretory globulin A secretory globulin A
  • hematopoietic stem cells which are essentially associated with T cell differentiation
  • preventing thymic involution or preventing the hypertrophy of mesenteric lymph nodes (MLNs) or spleens.
  • the recombinant protein of the present invention prevents, improves, or treats the deteriorations in metabolic functions, functions of skin tissues, functions of intestinal tissues, functions of muscular tissues, brain functions, or cardiovascular functions. According to another embodiment of the present invention, the recombinant protein of the present invention prevents, improves, or treats the deteriorations in metabolic functions, functions of skin tissues, functions of intestinal tissues, or functions of muscular tissues. According to a particular embodiment of the present invention, the recombinant protein of the present invention prevents, improves, or treats the deteriorations in functions of the dermal tissue in which hair follicles are present, functions of large intestine tissues, or functions of muscular tissues.
  • the present invention provides a composition for preventing, improving, or treating metabolic disease, the composition comprising the recombinant protein as an active ingredient.
  • the term “metabolic disease” refers to a syndrome in which risk factors, such as obesity, diabetes, hypertriglyceridemia, hypertension, cardiovascular disease, and blood clotting, are shown together.
  • the syndrome per se is not fatal, but has a predisposition to severe diseases, such as diabetes or ischemic cardiovascular diseases, resulting in a great threat to modern people.
  • the syndrome was once called several names, including syndrome X, since the cause of the syndrome was not known, but recently, the syndrome was officially named metabolic syndrome or insulin resistance syndrome through adult treatment program III (ATP III) established by the World Health Organization and the US National Institutes of Health.
  • the metabolic disease of the present invention is obesity.
  • the present invention provides a composition for preventing, improving, or treating hair-related disease, the composition comprising the recombinant protein as an active ingredient.
  • Examples of the hair-related disease which can be prevented, improved, or treated by the composition of the present invention include the reduction of hair gloss, hair decoloration, hair loss, the thickness reduction of the dermal layer in which hair follicles are present, the reduction in the number of follicles, and the like, but are not limited thereto.
  • the present invention provides a composition for preventing, improving, or treating bowel disease, the composition comprising the recombinant protein as an active ingredient.
  • Examples of the bowel disease which can be prevented, improved, or treated by the composition of the present invention include irritable bowel syndrome (IBS), uncontrolled diarrhea-associated irritable bowel syndrome (dIBS), Crohn's disease, traveler's diarrhea, ulcerative colitis, enteritis, small intestine bacterial overgrowth, chronic pancreatitis, pancreatic insufficiency, colitis, diverticular disease, hepatic encephalopathy, and hernia, but are not limited thereto.
  • the bowel disease of the present invention is enteritis or hernia.
  • the bowel disease of the present invention is colitis or hernia.
  • the present invention provides a composition for preventing, improving, or treating bone disease, the composition comprising the recombinant protein as an active ingredient.
  • the bone disease which can be prevented or treated by the composition of the present invention examples include osteoporosis, scoliosis, osteomalacia, rickets, bone metastasis of cancer cells, bone damage caused by bone metastases of cancer cells, osteolysis caused by bone metastases of cancer cells, fibrous dysplasia, aplastic bone disease, metabolic bone disease, rheumatoid arthritis, osteoarthritis, degenerative arthritis, and disc disease, but are not limited thereto.
  • the bone-related disease of the present invention is osteomalacia, metabolic bone disease, rheumatoid arthritis, osteoarthritis, or degenerative arthritis.
  • the bone disease of the present invention is osteoporosis or osteomalacia.
  • composition for preventing, improving, or treating the foregoing diseases of the present invention contains the foregoing recombinant protein as an active ingredient, and thus descriptions of overlapping contents with the recombinant protein are omitted to avoid excessive complication of the specification due to repetitive descriptions thereof.
  • composition of the present invention may be provided as a pharmaceutical composition.
  • the composition of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the recombinant protein any one of the recombinant proteins of the present invention; and (b) a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to an amount enough to show and accomplish efficacies and activities of the recombinant protein of this invention as described above.
  • the pharmaceutical composition may contain pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to.
  • the pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannitol, mannito
  • the pharmaceutical composition of this invention may be administered mucosally, orally, or parenterally, according to an embodiment, mucosally injection.
  • a suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition, and physicians of ordinary skill in the art can determine an effective amount of the pharmaceutical composition for desired treatment.
  • suitable dosage unit for human host is to administer with the pharmaceutical composition in 0.001-100 mg/kg (body weight).
  • the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form.
  • Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.
  • composition of the present invention may be provided as a food composition.
  • the composition of the present invention is a food composition
  • a food composition comprising (a) a sitologically effective amount of the recombinant protein any one of the recombinant proteins of the present invention; and (b) a sitologically acceptable carrier.
  • the food composition of the present disclosure may comprise, in addition to the recombinant protein of the present disclosure as active ingredients, ingredients commonly added for preparation of food.
  • proteins, carbohydrates, fats, nutrients, seasoning or flavors may be added.
  • the carbohydrate may be, for example, a sugar such as a monosaccharide, e.g. glucose, fructose, etc., a disaccharide, e.g.
  • the flavor may be a natural flavor [thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.]) or a synthetic flavor (saccharin, aspartame, etc.).
  • the food composition of the present disclosure when prepared as a drink, it may further comprise, in addition to the recombinant protein of the present disclosure as the active ingredient, citric acid, high-fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, eucommia extract, jujube extract, licorice extract, or the like.
  • composition of the present invention may be provided as a cosmetic composition.
  • the composition of the present invention contains not only the foregoing recombinant protein but also ingredients normally used in the cosmetic composition, for example, a carrier and normally additives, such as an antioxidant, a stabilizer, a solubilizer, vitamins, a pigment, and a flavor.
  • a carrier and normally additives such as an antioxidant, a stabilizer, a solubilizer, vitamins, a pigment, and a flavor.
  • purified water purified water, monohydric alcohol (ethanol or isopropyl alcohol), polyhydric alcohol (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acid (palmitic acid or linolenic acid), oil (wheat germ oil, camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avocado oil, soybean water-added lecithin or fatty acid glyceride) or the like may be used, but the carrier is not limited thereto.
  • a surfactant, a sterilizer, an antioxidant, a UV absorber, an anti-inflammatory agent, and a refreshing agent may be added as needed.
  • the surfactant may include one selected from the group consisting of polyoxyethylene, hardened castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, glyceryl monostearate, sorbitan monostearate, polyoxyethyelene monostearate, sorbitan, sucrose fatty acid ester, hexaglycerine monolaurate, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamate, isostearic acid, diester, N-acetylglutamine, and isostearyl ester.
  • the sterilizer may include one selected from the group consisting of hinoki thiol, triclosan, chlorhexidine gluconate, phenoxy ethanol, resorcin, isopropyl methyl phenol, azulene, salicylic acid, and zinc pyrithione.
  • any one of butyl hydroxyanisole, gallic acid, propyl gallate, and erythorbic acid may be used.
  • any one of benzophenones such as dihydroxy benzophenone, melanin, para-amino benzoic acid ethyl, para-dimethylamino benzoic acid 2-ethylhexyl ester, cynocite, para-methoxy cinnamic acid 2-ethylhexylester, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, and metal oxide microparticles may be used.
  • benzophenones such as dihydroxy benzophenone, melanin, para-amino benzoic acid ethyl, para-dimethylamino benzoic acid 2-ethylhexyl ester, cynocite, para-methoxy cinnamic acid 2-ethylhexylester, 2-(2-hydroxy-5-methylphenyl)benzotriazole, urocanic acid, and metal oxide microparticles may be used.
  • glycyrrhetinic acid dipotassium or allantoin may be used, and as the refreshing agent, capsicum tincture or 1-menthol may be used.
  • the dosage form of the composition is any dosage form that can blend the recombinant protein as an active ingredient
  • examples of the dosage form of cosmetics for preventing hair loss may include various forms of a sol, a gel, an emulsion, oil, wax, aerosol, and the like, such as hair tonic, hair cream, hair lotion, hair shampoo, hair rinse, hair conditioner, hair spray, hair aerosol, pomade, a powder, and a gel, but are not limited thereto.
  • the present invention provides a composition for preventing, improving, or treating aging, the composition comprising, as an active ingredient, a recombinant protein of flagellin, which is the constituent of Vibrio vulnificus flagella, fused with a pathogenic protein antigen.
  • the present invention provides a composition for preventing, improving, or treating metabolic disease, hair-related disease, eye disease, bowel disease, or bone disease, the composition comprising the recombinant protein as an active ingredient.
  • the present invention can improve external and internal aging-related malfunctions and enhance immunity by using the recombinant protein of the present invention.
  • the present invention can easily perform immunization through mucosal administration of the composition of the present invention.
  • FIG. 1 shows a schematic view with respect to an experiment schedule and an experiment scheme for immunizing aged mice.
  • FIG. 2 shows graphs illustrating measurement results of changes in food intake and body weight in aged mice according to the immunization with FlaB-PspA recombinant protein.
  • FIG. 3 shows morphological change results of aged mice through continuous immunization of an antigen.
  • FIG. 4 is an IgA reaction graph in aged mice by continuous immunization.
  • FIG. 5 illustrates histopathological findings with respect to the cutaneous histological change of aged mice by immunization with FlaB-PspA recombinant protein.
  • FIG. 6 shows the changes of hematopoietic stem cells in marrow cells of aged mice by immunization with FlaB-PspA recombinant protein.
  • FIG. 7 shows organs illustrating lymphatic system changes of aged mice by immunization with FlaB-PspA recombinant protein.
  • FIG. 8 shows the change in bone mineral density of aged mice by immunization with FlaB-PspA recombinant protein.
  • mice (C57BL/6J, aged at least 23 months) were intranasally immunized with purified protein and recombinant protein continuously at two-week intervals.
  • PspA protein and FlaB-PspA protein were purchased from the laboratory of professor Jun-haeng, Lee of the Clinical Vaccine Development Project Group of Chonnam National University.
  • mice All immunization experiments were conducted in the specific pathogen free (SPF) facility. Young mice (8-10 week old) were used for a control group. A non-immunized aged mouse group, an aged mouse group intranasally immunized with PBS (16 ⁇ l/mouse) 8 times or more at 2-week intervals, an aged group intranasally immunized with only PspA (2.5 ⁇ g/16 ⁇ l/mouse) protein 8 times or more at 2-week intervals, and an aged group intranasally immunized with FlaB-PspA (6.5 ⁇ g/16 ⁇ l/mouse) recombinant protein 8 times or more at 2-week intervals were used for immunization experimental groups.
  • PBS 16 ⁇ l/mouse
  • PspA 2.5 ⁇ g/16 ⁇ l/mouse
  • FlaB-PspA 6.5 ⁇ g/16 ⁇ l/mouse
  • mice The morphological and behavioral changes of the aged mice were observed during the continuous immunization.
  • mice were separately managed one by one.
  • 50 g of feed was provided for each mouse, and then the remainder of the feed was accurately measured at one-week intervals.
  • body weight of each mouse was accurately measured using an animal scale every week.
  • mice In order to compare morphological changes of aged mice according to the immunization, the appearances of the mice, that is, the hair condition, hair loss, and decoloration was observed and the anus or eyes were also continuously observed, thereby collecting changed patterns.
  • the behavioral changes of the mice were observed at a certain point during the immunization.
  • the aged mice of each group were placed in a confined space, and the motions of the mice were observed for a period of time. The motions were compared based on general standard items of mouse behavioral ability, that is, the motion, the number of times of standing on hind legs, the number of times of supporting using forelegs, and the number of times of touching the nose.
  • H&E staining In order to compare the morphological changes of the aged mice according to the immunization, various tissues including the skin tissue were collected, and fixed with formalin prior to making a paraffin-block, and pathological findings were compared through Hematoxylin and Eosin (H&E) staining. In addition, with reference to the effect of immunization and the feed intake through the mucosal immune response, pathological findings of the tissues of small and large intestines, which largely account for the mucosal immunity, were compared and observed through H&E staining.
  • the lymphocytes were separated from the cervical lymph nodes and spleen and then the antigen-mediated cellular response was compared by ELISA.
  • the bone marrow cells were separated from hind legs of the aged mice, and the comparison was conducted using flow cytometry (FACS, Beckman Coulter). Particularly, the frequency of hematopoietic stem cells essentially associated with T cell differentiation was compared and observed through immuno-staining using CD34, which is a hematopoietic stem cell indicator.
  • the morphological and behavioral changes of the aged mice according to the continuous immunization were compared and compared in connection with metabolism.
  • the serum or plasma was separated therefrom, and then the changes in hormone-related and metabolism-related genes were compared.
  • the intestine-associated microenvironment was compared using normal microbiota.
  • the blood and feces were collected from the aged mice continuously immunized with antigens, and then the gene expression pattern was compared and observed through advanced analysis methods, such as microarray.
  • mice While the aged mice were intranasally immunized eight times at two-week intervals, the body weight and the feed intake of the mice of each group were measured. The non-immunized aged mice were used as a control group.
  • mice were intranasally immunized eight times with phosphate buffered saline (PBS), prepared 2.5 ⁇ g of PspA, and 6.5 ⁇ g of FlaB-PspA recombinant protein at two-week intervals.
  • PBS phosphate buffered saline
  • FlaB-PspA recombinant protein The body weight and the feed intake of the mice of each group were measured every week. The measurement results are shown in FIGS. 2 a and 2 b.
  • the non-immunized mouse group showed no great change in the feed intake over time, and the group immunized with PBS also showed no great change in the feed intake, regardless of eight times of immunization at two-week intervals.
  • the group immunized with PspA alone was verified to show a gradual increase in the feed intake during the continuous immunization.
  • the group immunized with FlaB-PspA recombinant protein showed an increase in the feed intake through the continuous immunization, and here, the rate in increase of the feed intake was significantly higher than that of the group immunized with PspA alone ( FIG. 2 b ).
  • mice While the aged mice were intranasally immunized eight times at two-week intervals, the morphological changes of the mice according to the immunization were monitored every week. The results are shown in FIG. 3 .
  • the non-immunized aged mice were used as a control group.
  • mice prior to the immunization had no abnormal findings by appearances. There were no abnormal findings in view of hair condition, hair luster, hair decoloration, hair loss, the anus (colitis or hernia), or the eyes (cataracts).
  • the feces were collected from the mice of each group after each immunization to verify the IgA reaction of the aged mice according to the immunization by ELISA. The results are shown in FIG. 4 .
  • the non-immunized aged mice were used as a control group.
  • mice had no great difference in the IgA response during the immunization (O-control). It was verified that the group immunized with PBS alone showed no great difference in the IgA response, and then showed a slight increase in the IgA response after the sixth immunization, but there is no great increase in the IgA response (O-PBS). The group treated with PspA alone showed the IgA response after the fourth immunization, but showed no great difference after that (O-PspA). Whereas, as for the group immunized with FlaB-PspA recombinant protein, the IgA response was significantly increased depending on the number of times of immunization (O-FlaB-PspA).
  • the results of example 2 indicated that the hair condition of the aged mice became very favorable in the group continuously immunized with antigens, particularly, the recombinant protein.
  • the results were confirmed through H&E staining.
  • the H&E staining results are shown in FIG. 5 . Young mice were used as a control group.
  • the histological assay confirmed that the continuous immunization with antigens improved the morphological findings of the aged mice (results of example 2). It could be confirmed through the histological assay that the amelioration of the progression of hair decoloration and hair loss of the aged mice, shown in the results of example 2, is due to the fact that the hair follicles are maintained in the aged mice due to the continuous immunization. In view of the results, it can be seen that the continuous immunization with the recombinant protein slows or prevents the progression of hair decoloration and hair loss occurring due to aging.
  • CD34 which is a hematopoietic stem cell indicator
  • mice were extracted from the mice of each group, and the morphology and weight of each organ were measured. The measurement results are shown in FIG. 7 . Young mice were used as a control group.
  • the tissues were extracted from the aged mice, and the morphological change and weight of each of the organs were measured. As a result, the difference caused by the continuous immunization was observed in the lymphatic system-related organs rather than in the other organs. Particularly, it was verified that the morphology and weight of the organs in the group immunized with FlaB-PspA recombinant protein were similar to those of the young mice.
  • Thymic involution with age is a general phenomenon, but it was verified that the thymus weight was increased in the aged mouse groups continuously immunized with antigens, and particularly, the thymus weight was significantly increased in the group immunized with FlaB-PspA recombinant protein.
  • the aged mice continuously immunized with antigens exhibited improved morphological features of the immune-related organs.
  • the aged mice immunized with FlaB-PspA recombinant protein were observed to have very similar organ morphological findings to the young mice.

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US11261241B2 (en) 2008-05-23 2022-03-01 Siwa Corporation Methods, compositions and apparatuses for facilitating regeneration
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US11518801B1 (en) 2017-12-22 2022-12-06 Siwa Corporation Methods and compositions for treating diabetes and diabetic complications
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JP2021522271A (ja) * 2018-04-24 2021-08-30 ゲノム プロテクション，インコーポレイテッド 虚弱および加齢を改善するための方法
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