Vaccine delivery system O / W emulsion nanoparticles

Abstract

The present invention relates to a vaccine delivery system comprising: water-in-water (O / W) emulsion nanoparticles comprising carboxylated polyoxyethylene sorbitan ester, a surfactant of sorbitan ester, and squalene oil; and an antigen, wherein the emulsion nanoparticles and the antigen are chemically bound. The nanocomposite vaccine delivery system according to the present invention effectively delivers immune antigens to immune cells and can induce the activation of an immune response by effectively stimulating immune cells along with the immune-enhancing action of the emulsion particles. Through this, prevention against the corresponding antigen and enhancement of immunity can be expected.

Description

Technology Field

[0001] The present invention relates to a vaccine delivery vehicle comprising O / W emulsion nanoparticles and an antigen, and a vaccine composition comprising said vaccine delivery vehicle. Background Technology

[0003] Recently, outbreaks of various viruses, such as influenza and COVID-19, have caused social and economic losses worldwide every year. In this pandemic situation, the development of vaccine technology for virus prevention is crucial.

[0004] Generally, vaccines are classified into live vaccines, attenuated vaccines, inactivated vaccines, and recombinant vaccines. Live and attenuated vaccines require a long time to manufacture and have high production costs, and they also entail stability issues. Therefore, research and development of recombinant vaccines, which offer superior stability and production efficiency, is actively underway; however, recombinant vaccines have a critical drawback: lower immunogenicity compared to vaccines derived from existing viruses.

[0005] Existing recombinant protein antigens not only had the disadvantage of low immunogenicity when administered alone, but the antigen delivery ability of existing commercial adjuvants was also low.

[0006] Therefore, in a situation where the emergence of new variant viruses is continuous, there is a need for technology that enhances immunogenicity by applying it to newly developed recombinant proteins. Prior art literature

[0007] Korean Published Patent No. 2020-0048334 The problem to be solved

[0008] Accordingly, the inventors have completed the invention of a vaccine delivery vehicle capable of efficiently inducing immune cell activation by an antigen through the immune-enhancing effect of the emulsion by chemically combining an antigen with an O / W emulsion nanoparticle containing a carboxylated twin surfactant to produce a nanocomposite that enables improved delivery ability and simultaneous delivery of the particle and the antigen.

[0010] The objective of the present invention is to provide a vaccine delivery system with enhanced prevention and immunity against antigens.

[0011] Another objective of the present invention is to provide a vaccine composition comprising the vaccine delivery vehicle. means of solving the problem

[0013] The present invention will be described in detail below.

[0014] The present invention provides a vaccine delivery system comprising (a) oil-in-water (O / W) emulsion nanoparticles comprising carboxylated polyoxyethylene sorbitan ester, a surfactant of sorbitan ester, and squalene oil; (b) an antigen; and (c) an adjuvant, wherein the emulsion nanoparticles and the antigen are chemically bound.

[0015] The oil-in-water (O / W) emulsion nanoparticles according to the present invention can be formed by mixing a carboxylated polyoxyethylene sorbitan ester, a surfactant of the sorbitan ester, and squalene oil. For example, as shown in the attached drawing Fig. 1, the O / W emulsion nanoparticles have a hydrophobic core formed by squalene oil and a carboxylated surfactant distributed on the outside (shell). The hydrophobic portion of the carboxylated polyoxyethylene sorbitan ester and the squalene oil form a hydrophobic non-covalent bond, resulting in a particle shape with a hydrophilic exterior. The sorbitan ester acts as an auxiliary surfactant, promoting the self-assembly reaction through the bond and stabilizing the particles.

[0016] In the present invention, the polyoxyethylene sorbitan ester may be polysorbate 80 (tween 80), but is not limited thereto.

[0017] In the present invention, the sorbitan ester may be sorbitan trioleate (Span85), but is not limited thereto.

[0018] It is preferable that the concentration of the above polyoxyethylene sorbitan ester or sorbitan ester be 0.5% (w / v) to 0.8% (w / v) so that vaccine delivery particles can be composed of 100 to 500 nm, which is a size capable of efficiently penetrating into the cell through endocytosis.

[0019] The surfactant of the above O / W emulsion nanoparticles is a carboxylated polyoxyethylene sorbitan ester, and the sorbitan ester is mixed in a weight ratio of 0.5 to 1.5 : 0.5 to 1.5 because the promotion of the particle self-assembly mechanism and particle stabilization by the sorbitan ester are essential.

[0020] In the present invention, the squalene oil is preferably used in a concentration range of 2.5% (w / v) to 7.5% (w / v) for the purpose of preparing vaccine delivery particles with a size of 100 to 500 nm, which is ideal for efficient intracellular penetration at the surfactant concentration.

[0021] Accordingly, the above O / W emulsion nanoparticles can be prepared by mixing the surfactant and oil in a weight ratio of 1:3 to 15.

[0022] In one embodiment of the present invention, the O / W emulsion nanoparticles may have a diameter of 50 nm or more and 300 nm or less. Preferably, they may have a diameter of 100 nm or more and 290 nm or less, 110 nm or more and 290 nm or less, 120 nm or more and 290 nm or less, 130 nm or more and 290 nm or less, and 140 nm or more and 290 nm or less, but are not limited thereto.

[0023] In the present invention, the term "antigen" refers to a molecule or combination of molecules intended to induce an immune response so that the immune system of a living animal recognizes it.

[0024] In the present invention, the antigen may be selected from the group consisting of peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells, and cell fragments. The protein may include, for example, recombinant proteins, subunits, and split protein antigens, and the cell may include, for example, dendritic cells.

[0025] In the present invention, the antigen is OVA (ovalbumin), an antigen of coronavirus, an antigen of Japanese encephalitis virus, an antigen of HIB (Heamophilus influenzae type B), an antigen of Zika virus, an antigen of Pseudomonas aeruginosa, a pertussis antigen, an antigen of Mycobacterium tuberculosis, an antigen of Bacillus anthracnose, an antigen of HAV (Hepatitis A virus), an antigen of HBV (Hepatitis B virus), an antigen of HCV (Hepatitis C virus), an antigen of HIV (human immunodeficiency virus), an antigen of HSV (Herpes simplex virus), an antigen of Neisseria meningitidis, an antigen of Corynebacterium diphtheria, an antigen of Bordetella pertussis, an antigen of Clostridium tetani, It may be selected from the group consisting of antigens of HPV (human papilloma virus), varicella virus, enterococci, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Enterobacter, Helicobacter pylori, malaria, dengue virus, Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome Bunyavirus (SFTS Bunyavirus), influenza virus, Ebola virus, and pneumococcus.

[0026] The term "OVA (ovalbumin)" in this specification is referred to as ovalbumin, egg albumin, egg white albumin, etc., and is known as a major antigen protein that causes egg allergy.

[0027] The term “influenza virus” in this specification refers to an RNA virus belonging to the family Orthomyxoviridae, which is classified into three types: A, B, or C. Among these, type A has been confirmed to be infective in mammals such as humans and pigs, as well as various types of poultry and wild birds. The normal natural host of type A influenza virus is known to be wild waterfowl such as ducks. However, epidemiological studies on influenza infection in wild birds have confirmed that 16 types of HA and 9 types of NA influenza viruses are infecting wild birds.

[0028] The antigen of influenza virus type A can be H1N1, H1N2, H2N2, H3N1, H3N2, H5N1, or H5N8.

[0029] The above coronavirus may be selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, and specifically may be porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), or a combination thereof. SARS-CoV-2 may be the primary causative agent of Coronavirus Disease 2019 (COVID-19).

[0030] In the vaccine delivery vehicle of the present invention, the antigen may be in a form chemically bonded to emulsion nanoparticles. In particular, the antigen is distributed on the surface of the nanoparticles by binding to carboxyl groups distributed in the hydrophilic portion of carboxylated polyoxyethylene sorbitan ester.

[0031] At this time, it is preferable that the carboxyl group and the antigen included in the O / W emulsion nanoparticles be mixed in a molar ratio of 10 to 200:1, 20 to 150:1, or 30 to 100:1.

[0032] When administered in this form, unlike conventional particles that are delivered solely through the simple adsorption of a single antigen or emulsion particles, it is possible to efficiently deliver both the adjuvant emulsion nanoparticles and the antigen to antigen-presenting cells. Furthermore, vaccine delivery particles ranging in size from 100 to 500 nm exhibit a higher delivery rate compared to antigens smaller than 10 nm through the endocytosis pathway.

[0033] Therefore, when utilizing the vaccine delivery vehicle of the present invention, there is an advantage that the antigen delivery rate can be maximized.

[0034] In the present invention, the term “chemical bond” means a covalent bond between the carboxyl group of the carboxylated polyoxyethylene sorbitan ester distributed on the surface of the emulsion nanoparticles and the amine group of the antigen.

[0035] In the present invention, the term "adjuvant" refers to a substance called an immunoadjuvant, an antigen enhancer, or an adjuvant, and more specifically, refers to a substance that helps the immune system produce a large amount of antibodies by enhancing the immune response to a vaccine. The said adjuvant may act through various mechanisms, such as one or more mechanisms including increasing the biological or immunological half-life of an antigen; improving antigen delivery to an antigen-presenting sequence; improving antigen processing and presentation by antigen-presenting cells; and inducing the production of immunomodulatory cytokines.

[0036] According to an embodiment of the present invention, the antigen delivery ability of a vaccine delivery vehicle containing the nanoparticles can be increased by 50 times or more, 40 times or more, 30 times or more, or 20 times or more compared to a vaccine delivery vehicle not containing the nanoparticles.

[0037] In one embodiment of the present invention, the vaccine delivery vehicle, which is a nanoparticle to which the antigen is bound, may have a diameter of 100 nm or more and 500 nm or less. Preferably, it may have a diameter of 100 nm or more and 450 nm or less, 150 nm or more and 450 nm or less, 100 nm or more and 400 nm or less, 150 nm or more and 400 nm or less, or 150 nm or more and 350 nm or less, but is not limited thereto.

[0038] The present invention provides a vaccine composition comprising a vaccine delivery vehicle and a pharmaceutically acceptable carrier.

[0039] All the contents described above regarding vaccine delivery vehicles may be applied or adopted by analogy to vaccine compositions.

[0040] In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that imparts immunity to a living organism, and means an immunogen or antigenic substance that induces immunity in a living organism by administering it to a person or animal for the prevention of infection and / or infectious diseases.

[0041] The above vaccine may be in the form of an inactivated vaccine, an attenuated vaccine, a subunit vaccine, a conjugate vaccine, a recombinant vaccine, a monovalent vaccine, a multivalent vaccine, or a combination vaccine.

[0042] In the present invention, the vaccine may be intended to prevent or treat infection or infectious disease caused by bacteria and / or viruses.

[0043] In the present invention, the term "prevention" refers to any act of suppressing or delaying infection by bacteria and / or viruses and the onset of disease caused by said infection through the administration of said vaccine composition.

[0044] In the present invention, the term "treatment" refers to any act in which the symptoms of a disease already induced by bacterial and / or viral infection are improved or benefited by the administration of the vaccine composition.

[0045] "Pharmaceuticalally acceptable carriers" include any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antimicrobial and antifungal agents, isotonic agents, adsorption retardants, etc. Carriers, excipients, and diluents that may be included in a vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0046] In addition, the vaccine composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injectable solutions, according to conventional methods. When formulating, the composition may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, and these solid formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the lecithin-like emulsifier. In addition, lubricants such as magnesium styrate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration may include suspensions, oral liquids, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for non-oral administration include sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, and lyophilized preparations. Water-insoluble preparations and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0047] Administration of the present invention means introducing a specific substance to a subject by any appropriate method, and the route of administration may be administered through any general route that can reach the target tissue. For example, it may be administered intraperitoneally, intramuscularly, orally, subcutaneously, topically, or by immersion, but is not limited thereto.

[0048] The preferred dosage of the above vaccine composition may vary depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the adjuvant of the present invention may be administered at a dose of 10 to 100 mg / kg per day, preferably 50 to 500 mg / kg. The administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way. The above dosage may be determined based on factors including the type and severity of the individual's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0050] In addition, the present invention provides a method for preventing or treating infections and infectious diseases caused by bacteria, viruses, and / or microorganisms, comprising the step of administering the vaccine composition to an individual. All details described above regarding the vaccine delivery vehicle and the vaccine composition may be applied or mutatis mutandis to the above method.

[0051] In the present invention, the term "individual" may include, without limitation, animals such as rats, livestock, mammals including humans, farmed fish, etc., that have developed or are at risk of developing an infection or infectious disease caused by bacteria, viruses, and / or microorganisms. The individual may exclude humans.

[0052] The above vaccine composition may be administered as a single or multiple doses in pharmaceutically effective amounts. In this case, the composition may be administered in the form of a liquid, powder, aerosol, injection, intravenous fluid (Ringer), capsule, pill, tablet, suppository, or patch.

[0053] The route of administration of a vaccine composition for preventing or treating infections and infectious diseases caused by the above bacteria, viruses, and / or microorganisms may be any general route as long as it can reach the target tissue.

[0054] The above vaccine composition may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, nasal administration, pulmonary administration, and rectal administration, depending on the purpose. However, when administering orally, it may be administered in an unformulated form, and since the vaccine composition may be denatured or degraded by gastric acid, the oral composition may be administered into the mouth in a form coated with an active agent or formulated to protect against degradation in the stomach, or in the form of an oral patch. Additionally, the above composition may be administered by any device capable of transporting the active substance to target cells. Effects of the invention

[0056] The nanocomposite vaccine delivery system according to the present invention effectively delivers immune antigens to immune cells and can induce the activation of an immune response by effectively stimulating immune cells along with the immune-enhancing action of the emulsion particles. Through this, prevention against the corresponding antigen and enhancement of immunity can be expected. Brief explanation of the drawing

[0058] Figure 1 shows the structure of emulsion nanoparticles used as an adjuvant of the present invention and the structure of a vaccine delivery vehicle bound to an antigen. Figure 2 shows the synthesis process of carboxylated Tween 80 (CT80), a functional surfactant. Figure 3a shows the results of CT80 synthesis confirmed through FT-IR analysis. Figure 3b shows the results of CT80 synthesis confirmed through NMR analysis. Figure 4 shows the particle size of CTSq. Figure 5 shows the particle morphology analysis of CTSq using a transmission electron microscope. Figure 6 analyzes the stability and recovery of CTSq particles during the freeze-drying process. Figure 7 shows the results of particle size analysis of a CTSq / OVA nanocomposite in which nanoparticles CTSq and model antigen OVA were chemically combined using the EDC / NHS method. Figure 8 shows the morphology of the CTSq / OVA nanocomposite analyzed through a transmission electron microscope. Figure 9 shows the long-term stability of the CTSq / OVA nanocomposite at 4°C. Figure 10 shows the binding of antigen-binding particles (CTSq / OVA) prepared through agarose gel electrophoresis (a) and immunogold staining (b). Fig. 11 shows a single antigen (OVA) and The delivery ability of the CTSq / OVA nanocomposite antigen model to macrophages (a) and dendritic cells (b) was confirmed through flow cytometry. Figure 12 shows the intracellular delivery ability to the RAW264.7 (macrophage) cell line confirmed using a confocal microscope. Figure 13 shows the intracellular delivery ability to Mouse BMDC (dendritic cell) primary cells using a confocal microscope. Figure 14 shows the antibody production in mice after treatment with each sample for Total IgG (a), isotype IgG1 (b), and IgG2a (c) [NC: negative control, OVA: antigen alone, CTSq / OVA: chemical binding of CTSq and antigen, CTSq+OVA: simple mixture of CTSq and antigen, addavax: commercial adjuvant, addavax+OVA: simple mixture of addavax and antigen]. Figure 15 shows the antibody production ability on Day 14 and 28 after treating mice with each sample in Total IgG [NC: Negative control, Ag: Antigen alone, Alum + Ag: Simple mixture of commercial adjuvant Alum and antigen, CTSq / Ag: Chemical binding of CTSq and antigen]. Specific details for implementing the invention

[0059] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0061] [Example]

[0062] Example 1: Synthesis and Analysis of CT80 (Carboxylated Tween80)

[0063] 1) Synthesis of carboxyl-modified functional surfactant (CT80)

[0064] A functional surfactant modified with a carboxyl group (Carboxylated Tween80, CT80) was synthesized via a carboxylation reaction of Tween80 (Fig. 1). 3 g of Tween80 was dissolved and mixed in 100 g of 1,4-dioxane, and then substances were added such that the molar ratio of Tween80:succinic anhydride (SA):4-dimethylaminopyridine (DMAP):triethylamine (TEA) was 1:4:4:4. The reaction was then carried out by stirring at 25 °C for 48 hours. After the reaction was completed, 1,4-dioxane was evaporated by vacuum distillation in a 60 °C water bath, and then 10 mL of carbon tetrachloride was mixed in. The mixture was re-mixed with 40 mL of distilled water and stirred at 25 °C for 48 hours to remove residual organic solvent. Afterward, impurities were removed by dialyzing in distilled water for 4 days using a dialysis membrane (molecular weight cut off: 1 kDa), and then freeze-dried to obtain the final product, carboxylated Tween80.

[0065] 2) FT-IR Analysis

[0066] The results of CT80 synthesis were confirmed through Fourier Transform Infrared Spectroscopy (FT-IR) analysis and are shown in Figure 3a.

[0067] When examining the typical FT-IR profiles of the Tween80 and CT80, at 1550 nm -1 and 1640nm -1 The peak of the carboxyl group present in was clearly observed, confirming that the carboxylation of CT80 was successfully achieved.

[0068] 3) NMR analysis

[0069] To confirm more accurately, the results of CT80 synthesis were verified through Nuclear Magnetic Resonance (NMR) analysis and are shown in Figure 3b.

[0070] When examining the H-NMR spectra of general Tween80 and CT80, a (-CH2CH2-) peak specifically occurred around 2.6 ppm in the CT80 spectrum, further confirming that the carboxylation of CT80 was successfully achieved.

[0072] Example 2: Preparation of O / W emulsion nanoparticles (CTSq) containing CT80

[0073] 1) Preparation and Analysis of Nanoparticles (CTSq) According to Surfactant Ratio

[0074] To prepare O / W emulsion nanoparticles (CTSq) containing CT80, Span 85, a substance capable of acting as a squalene oil co-surfactant, was used. CT80 was added to DPBS buffer (pH 7.4) at a specified concentration (0.5 × 10⁻⁶). -3 It was added to achieve a concentration of %(w / v) ~ 0.8 %(w / v)). The mixed solution was vigorously stirred in a stirrer for 3 minutes. At the same concentration as CT80 (0.5×10⁻⁶ -3 Span 85 (%(w / v) ~ 0.8%(w / v)) was dissolved in THF (tetrahydrofuran). The amount of squalene oil was determined to be a fixed concentration (2.5%(w / v) ~ 7.5%(w / v)) relative to the solvent (DPBS) of the mixture being stirred.

[0075] An oil phase solution was prepared by mixing squalene oil with Span 85 dissolved in THF. The oil phase solution was added to the stirring mixture, and sonication was performed immediately for 15 minutes using a tip sonicator. After the synthesis was completed, the mixture was stirred overnight at 25°C to remove residual organic solvent. After the O / W emulsion nanoparticles were prepared, particle size analysis was performed using an ELSZ-2000 (Otsuka Electronics) and is shown in Figure 4.

[0076] It was confirmed that uniform and stable particles with a size of 300 nm or less were produced when the surfactant ratio to the squalene oil concentration (4.3 % (w / v)) was 0.5 % (w / v) or higher. The following experiment was conducted using O / W emulsion nanoparticles (CTSq) prepared at a surfactant ratio of 0.8 % (w / v), which is the condition for achieving the desired size and uniformity.

[0077] The shape and size of the nanoparticles were confirmed using TEM (Transmission electron microscopy, JEOL-F200) and are shown in Figure 5.

[0078] It was confirmed that the particles have a spherical shape and a uniform size.

[0079] 2) Evaluation of recovery after freeze-drying

[0080] A lyophilization recovery assay was performed to evaluate the recovery of the prepared O / W emulsion nanoparticles (CTSq) after freeze-drying. 1 mL of the particles were freeze-dried for 72 hours to solidify them, redispersed in 1 mL of distilled water, and then analyzed for particle size using DLS, as shown in Figure 6. Although the particles did not fully recover immediately after redispersion, it was confirmed that the particles recovered to a size and dispersion similar to the original particles after sonication.

[0081] 3) Synthesis of antigen-binding particles (CTSq / OVA) through chemical binding of O / W emulsion nanoparticles (CTSq) and protein antigens (Ovalbumin, OVA)

[0082] The EDC / NHS coupling method was used for the binding of CTSq with the model antigen OVA (ovalbumin). For CTSq / NHS modification, 4 μmol EDC (ethyl(dimethylaminopropyl)carbodiimide) and 8 μmol NHS (N-Hydroxysuccinimide) dissolved in MES buffer (pH 5.3) were added to 25 μl of CTSq (mol of -COOH: 0.4 μmol) to achieve a CTSq:EDC:NHS molar ratio of 1:10:20, and the mixture was vigorously stirred at 25 °C for 1 hour. Subsequently, to prevent non-specific binding between antigens and particle aggregation, 128 μmol 2-ME (2-mercaptoethanol), an EDC deactivator, was added and the mixture was stirred for 15 minutes. Particles were prepared by adding 8 nmol OVA such that the molar ratio of the carboxyl groups in CTSq to the antigen OVA was 50:1 and stirring for 2 hours. The prepared particles were dialyzed over PBS buffer (pH 7.4) for 18 hours using a dialysis membrane (molecular weight cut off: 100 kDa) and stored at 4 ℃ for the next experiment.

[0083] 4) Analysis of antigen-binding particles (CTSq / OVA)

[0084] Antigen-binding particles (CTSq / OVA) were prepared by chemically binding the antigen OVA to O / W emulsion nanoparticles (CTSq).

[0085] The prepared particles were analyzed using ELSZ-2000 (Otsuka Electronics) and TEM (JEOL-F200) and are shown in Figures 7 and 8. It was confirmed that the size increased as the antigen bound to the nanoparticles, and that the zeta potential increased as the carboxyl groups distributed on the surface of the emulsion nanoparticles were consumed by antigen binding. TEM images confirmed that the antigen-bound particles existed in a single particle form and had a uniform size. To analyze the long-term stability of the antigen-bound particles, the particle size distribution was measured for 56 days and is shown in Figure 9. It was confirmed that the particles remained stable for at least 56 days when stored at 4°C.

[0086] To confirm the chemical binding of the prepared antigen-binding particles (CTSq / OVA), agarose gel electrophoresis and immunogold staining were performed and are shown in Figure 10.

[0087] In agarose gel electrophoresis experiments, three groups were tested: i) single antigen (OVA), ii) antigen-bound particles (CTSq / OVA), and iii) a simple mixture of antigen and particles (CTSq + OVA). Electrophoresis was performed on a 1% agarose gel at 100V for 60 minutes, followed by protein staining with Sun Gel Staining Solution, and the results were verified using the GelDoc Go Gel Imaging System. While OVA bands were observed in the single antigen treatment and the simple mixture group, the absence of an OVA band in the antigen-bound particles group and the staining of the top of the wells confirmed that chemical binding between the particles and the antigen was well established.

[0088] Immunogold staining was performed on two groups: i) basic particles (CTSq) and ii) antigen-bound particles (CTSq / OVA). Samples mounted on a TEM grid were treated with serum containing an OVA-specific polyclonal antibody diluted 250-fold in DPBS, followed by treatment with an AuNP-conjugated mouse IgG-specific goat antibody, and analyzed using a TEM (JEOL-F200). It was further confirmed that chemical binding occurred normally, as AuNPs were distributed at a high rate around CTSq / OVA, whereas this was not the case for CTSq.

[0090] Example 3: Confirmation of Cell-Level Antigen Delivery Ability of CTSq / OVA

[0091] To confirm delivery capacity through flow cytometry and confocal microscopy, model antigen OVA labeled with Alexa Flour 488 (AF-488) fluorescence was used.

[0092] Experiments were conducted under the following five conditions, and the immunoantigen model OVA-AF488 was diluted to 5 µg for the experiment.

[0093] i) Negative control (PBS buffer),

[0094] ii) Fluorescently labeled monoantigen (OVA-AF488),

[0095] iii) Antigen (chemical) bound particle (CTSq / OVA-AF488),

[0096] iv) Simple mixture of antigen and particle (CTSq + OVA-AF488),

[0097] v) Simple mixture of antigen and commercial adjuvant (Addavax + OVA-AF488)

[0098] The delivery capacity to RAW264.7 (Macrophage) cell line and Mouse BMDC (Dendritic Cell) primary cells under the above five conditions was quantitatively analyzed by flow cytometry and is shown in Figure 11. It was confirmed that when compared to a sample of a simple mixture of a single antigen and antigen particles targeting macrophages, the antigen delivery capacity of the antigen-binding particles increased by more than 15.3 times, and when compared to dendritic cells under the same conditions, the delivery capacity increased by more than 16.3 times.

[0099] The delivery ability to RAW264.7 (macrophage) cell line and Mouse BMDC (dendritic cell) primary cells under the above five conditions was visualized using a confocal microscope and is shown in Figures 12 and 13. Compared to the fact that OVA-AF488 (abstract) showed almost no fluorescence when treated with other samples, strong fluorescence values ​​confirmed that the model antigen was highly distributed in the cytoplasm of the antigen-binding particle (CTSq / OVA-AF488).

[0100] In other words, it was quantitatively and qualitatively confirmed that the ability to deliver antigens into cells was enhanced when delivered as antigen-binding particles.

[0102] Example 5: Confirmation of antibody production ability in an animal model

[0103] Mice (BALB / c) were administered two injections at 2-week intervals for intramuscular injection, and blood was collected at weeks 2 and 4 to measure immune activity using ELISA to confirm the antigen-specific antibody-based immune response (humoral immunity).

[0104] Immunological activity in mice was measured by diluting collected serum to a volume of 1:128 and confirming the amounts of IgG and its isotypes (IgG1, IgG2a) using ELISA (Enzyme-linked immunosorbent assay).

[0105] Animal experiments were conducted under five conditions: i) Negative control (PBS buffer), ii) single antigen (OVA), iii) antigen-binding particles (CTSq / OVA), iv) simple mixture of antigen and particles (CTSq + OVA), and v) simple mixture of antigen and commercial adjuvant (Addavax + OVA). The amount of antigen used was 10 µg / injection. Five mice were used for each condition, and the model antigen OVA was used as the immunization vaccine antigen.

[0106] As a result of conducting an ELISA experiment to determine the amounts of IgG and IgG1 at absorbance (450 nm), it was confirmed that the experimental group (CTSq / OVA) produced the highest amount compared to other control groups. In the case of IgG2a, a similar amount of antibody was produced as that of a commercial adjuvant and antigen mixed sample, but a significant difference occurred when compared to a single antigen (Fig. 14).

[0108] Example 6: Confirmation of antibody production ability in an animal model using other antigens

[0109] CTSq / Ag was prepared by applying a weakened influenza H3N2 virus (hereinafter referred to as Ag) to the O / W emulsion nano-adjuvant developed in this technology.

[0110] Mice (BALB / c) were administered two injections at 2-week intervals for intramuscular injection, and blood samples were collected at weeks 2 and 4 to measure immune activity using ELISA in order to confirm the antigen-specific antibody-based immune response (humoral immunity). The immune activity of the mice was measured by diluting the collected serum to a volume of 1:128 and determining the amounts of IgG and its isotypes (IgG1, IgG2a) using ELISA (Enzyme-linked immunosorbent assay).

[0111] Animal experiments were conducted using five conditions: i) negative control (PBS buffer), ii) single antigen (Ag), iii) a simple mixture of commercial adjuvant and antigen (Ag + Alum), and iv) chemically bound particles of CTSq and antigen (CTSq / Ag). Five mice were used for each condition, and the weakened cH3N2 virus (Ag) was used as the immunization vaccine antigen.

[0112] As a result of conducting an ELISA experiment to determine the amount of IgG at absorbance (450 nm), it was confirmed that the experimental group (CTSq / Ag) at week 4 of vaccine administration had an effect similar to that of Alum, which is currently commercially available and in use. In addition, a significant difference in antibody production occurred when compared to when a single antigen was administered. When comparing antibody production at day 14 and day 28, antibody production was significantly higher compared to other control groups, confirming that the vaccine formulation developed using this technology has a boosting effect [Fig. 15].

[0114] Although the present invention has been described with reference to the above-described embodiments and experimental examples, this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent alternative embodiments and experimental examples are possible therefrom. Accordingly, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

Claim 1 A vaccine delivery system comprising: a surfactant comprising carboxylated polysorbate 80 (Tween 80) and sorbitan trioleate (Span85); water-in-oil (O / W) emulsion nanoparticles comprising squalene oil; and an antigen comprising an amine group, wherein the emulsion nanoparticles and the antigen are covalently bonded. Claim 2 delete Claim 3 delete Claim 4 In claim 1, the surfactant of the nanoparticles is a vaccine delivery vehicle in which carboxylated polysorbate 80 and sorbitan trioleate (Span 85) are mixed in a weight ratio of 0.5 to 1.5 : 0.5 to 1.

5. Claim 5 A vaccine delivery vehicle according to claim 1, wherein the surfactant of the nanoparticles has a concentration of 0.5% (w / v) to 0.8% (w / v). Claim 6 A vaccine delivery vehicle according to claim 1, wherein the squalene oil of the nanoparticles has a concentration of 2.5% (w / v) to 7.5% (w / v). Claim 7 delete Claim 8 In claim 1, the antigen containing the amine group is OVA (ovalbumin), an antigen of coronavirus, an antigen of Japanese encephalitis virus, an antigen of HIB (Heamophilus influenzae type B), an antigen of Zika virus, an antigen of Pseudomonas aeruginosa, pertussis antigen, Mycobacterium tuberculosis antigen, anthracnose antigen, an antigen of HAV (Hepatitis A virus), an antigen of HBV (Hepatitis B virus), an antigen of HCV (Hepatitis C virus), an antigen of HIV (human immunodeficiency virus), an antigen of HSV (Herpes simplex virus), an antigen of Neisseria meningitidis, an antigen of Corynebacterium diphtheria, an antigen of Bordetella pertussis, and Clostridium tetani Antigen of tetani), antigen of HPV (human papilloma virus), antigen of Varicella virus, antigen of Enterococci, antigen of Staphylococcus aureus, antigen of Klebsiella pneumonia, antigen of Acinetobacter baumannii, antigen of Enterobacter, antigen of Helicobacter pylori, antigen of malaria, antigen of dengue virus, antigen of Orientia tsutsugamushi, severe fever with thrombocytopenia syndrome (Bunyavirus);A vaccine delivery vehicle selected from the group consisting of antigens of SFTS (Severe Fever with Thrombocytopenia Syndrome) virus, influenza virus, Ebola virus, and Streptococcus pneumoniae.; Claim 9 A vaccine delivery vehicle according to claim 1, wherein the mixing ratio of the carboxyl group and the antigen included in the nanoparticle is a molar ratio of 10 to 200:

1. Claim 10 A vaccine delivery vehicle according to claim 1, wherein the nanoparticles have an average diameter of 50 nm to 300 nm. Claim 11 In claim 1, the vaccine delivery vehicle has an average diameter of 100 nm to 500 nm. Claim 12 A vaccine composition comprising a vaccine delivery vehicle of claim 1 and a pharmaceutically acceptable carrier.

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