VACCINE ADJUVANT COMPOSITION BASED ON AMPHIPHILIC POLYAMINO ACID POLYMER, CONTAINING SQUALENE

Abstract

The present invention relates to an immunoadjuvant composition that includes amphiphilic polymer particles in an oil-in-water emulsion state containing an amphiphilic poly-amino acid polymer and a method of preparing the same. Since the immunoadjuvant composition of the present invention is prepared using an amphiphilic poly-amino acid polymer, it allows the provision of a vaccine immunoadjuvant composition which is more biocompatible and has a high antibody titer.

Claims

1. An amphiphilic poly-amino acid polymer represented by Formula 1 below: ##STR00006## Wherein R.sub.1 is one selected from the group consisting of ##STR00007## and x, y, and z are integers greater than 1.

2. The amphiphilic poly-amino acid polymer according to claim 1, wherein ##STR00008## the R.sub.1 is ##STR00009## and the R.sub.2 is ##STR00010##

3. An immunoadjuvant composition, comprising squalene-carrying amphiphilic polymer particles in an oil-in-water emulsion state which contain the amphiphilic poly-amino acid polymer according to claim 1.

4. The immunoadjuvant composition according to claim 3, wherein the composition comprises squalene in an amount of 0.5% (v/v) to 10% (v/v).

5. The immunoadjuvant composition according to claim 3, wherein a molar ratio of the squalene to the amphiphilic poly-amino acid polymer is 1:0.001 to 1:0.999.

6. A vaccine composition, comprising an antigen; and squalene-carrying amphiphilic polymer particles in an oil-in-water emulsion state which contain the amphiphilic poly-amino acid polymer according to claim 1.

7. The vaccine composition according to claim 6, wherein the antigen is an influenza virus antigen or a porcine epidemic diarrhea virus antigen.

8. A method of preparing an amphiphilic poly-amino acid polymer, the method comprising a step of reacting a compound represented by Formula 2 below with a compound represented by Formula 3 below to prepare an amphiphilic poly-amino acid polymer represented by Formula 1: ##STR00011## wherein, in Formulas 1 to 3, R.sub.1 is one selected from the group consisting of ##STR00012## and x, y, z, and p are integers greater than 1.

9. The method according to claim 8, wherein, in Formula 1, a ratio of x to y is 1:0.1 to 1:10.

10. A method of preparing an immunoadjuvant composition, the method comprising a step of mixing an aqueous solution comprising the amphiphilic poly-amino acid polymer according to claim 1 with a solution comprising squalene to prepare amphiphilic polymer particles in an oil-in-water emulsion state.

11. A method of preparing a vaccine composition, the method comprising a step of mixing a solution comprising an antigen with an emulsion solution comprising the squalene-carrying amphiphilic poly-amino acid polymer according to claim 1 which carries squalene.

12. The method according to claim 11, wherein, in the step, the solution comprising the antigen and the emulsion solution are mixed in a volumetric ratio of 1:0.2 to 1:5.

Description

DESCRIPTION OF DRAWINGS

[0046] FIG. 1 illustrates a synthesis process of an amphiphilic poly-amino acid polymer (PLL-b-Phe).

[0047] FIG. 2 illustrates a TEM image of an oil-in-water emulsion.

[0048] FIG. 3 illustrates a diagrammatic view of an oil-in-water emulsion.

[0049] FIG. 4 is a diagrammatic view illustrating a preparation method of an oil-in-water emulsion.

[0050] FIG. 5 illustrates a result for confirming a polymerization result of polylysine (PLL) through NMR analysis.

[0051] FIG. 6 illustrates a result for confirming a polymerization result of an amphiphilic poly-amino acid polymer (PLL-b-Phe) through NMR analysis.

[0052] FIG. 7 is a diagrammatic view illustrating a polymerization process of an amphiphilic poly-amino acid polymer (PLL-b-Phe).

[0053] FIG. 8 illustrates a results of emulsion size change dependent upon the amount of amphiphilic poly-amino acid polymer (PLL-b-Phe)-carried squalene (1%/200 l) investigated by a DLS method.

[0054] FIG. 9 illustrates a results of an emulsion size change dependent upon the amount of amphiphilic poly-amino acid polymer (PLL-b-Phe)-carried squalene (5%/1 ml) investigated by a DLS method.

[0055] FIG. 10 illustrates results of emulsion size change dependent upon the amount of an amphiphilic poly-amino acid polymer (PLL-b-Phe)-carried squalene (10%/2 ml) investigated by a DLS method.

[0056] FIG. 11 illustrates HI titer values measured through mouse erythrocyte aggregation inhibition experiments conducted to measure an antibody titer of a vaccine composition of the present invention under the following five conditions: (i) PBS (non-vac), ii) an influenza vaccine antigen (Ag-only), iii) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant for research (AddaVax), iv) a combination of an influenza vaccine antigen and PLL-b-Phe (Amino-Sq), and v) a combination of an influenza vaccine antigen and an addavax produced in a laboratory (T80-Sq)).

[0057] FIG. 12 illustrates a set of results for confirming the stability of particles dependent upon a storage temperature and period of amphiphilic amino acid polymer particles in an oil-in-water emulsion state.

[0058] FIG. 13 illustrates an ELISA result of IgG of serum collected from each of mouse models to measure the immunological activity of a vaccine composition of the present invention.

[0059] FIG. 14 illustrates an ELISA result of IgG1 of serum collected from each of mouse models to measure the immunological activity of a vaccine composition of the present invention.

[0060] FIG. 15 illustrates an ELISA result of IgG2a of serum collected from each of mouse models to measure the immunological activity of a vaccine composition of the present invention.

[0061] FIG. 16 illustrates weight measurement results of mouse models challenge-inoculated with an influenza virus (H1N1) to evaluate an infection inhibition ability of a vaccine composition according to the present invention.

[0062] FIG. 17 illustrates an antibody titer measurement result of virus collected from the lungs of each of mouse models at the 5th and 7th days of influenza virus infection.

[0063] FIG. 18 illustrates an ELISA result of IgG of serum collected from each of guinea pig models to measure the immunological activity of a vaccine composition according to the present invention.

BEST MODE

[0064] Hereinafter, the present application will be described in more detail with reference to the following examples. These examples are provided for illustrative purposes only and should not be construed as limiting the scope and spirit of the present application.

[Example 1] Preparation of Emulsion Including Squalene-Carrying Amphiphilic Poly-Amino Acid Polymer (PLL-b-Phe)

[0065] 1) Synthesis of Amphiphilic Poly-Amino Acid Polymer (PLL-b-Phe)

[0066] To polymerize a branched amphiphilic poly-amino acid polymer (PLL-b-Phe) of phenylalanine-NCA (Phe-NCA) which is an anhydrous hydrophobic amino acid with polylysine (Poly-Lys) which is a cationic poly-amino acid polymer, a polymerization degree (n) of Poly-Lys was found, and the masses thereof were determined such that a molar ratio of an amine group (NH.sub.2) of Poly-Lys to phenylalanine-NCA (Phe-NCA) became 1:0.9 to 0.1. The samples were dissolved in 25 mL of dimethylformamide (DMF) to prepare a solution mixture, followed by performing polymerization at 35 C. for 48 hours. A polymer was isolated from the reaction-terminated solution mixture using ethyl ether by a precipitation method, and was sufficiently dialyzed to remove the excess solvent and samples therefrom, and then lyophilized. As a result, an amphiphilic poly-amino acid polymer (PLL-b-Phe), in which Poly-Lys and Phe-NCA were polymerized in a branched form, was obtained. A synthesis process of PLL-b-Phe is illustrated in FIG. 1.

[0067] 2) Preparation of Oil-in-Water Emulsion

[0068] 40 mg of the amphiphilic poly-amino acid polymer (PLL-b-Phe) obtained in step 1) was dissolved in 20 mL of distilled water (DW), thereby preparing an aqueous solution. The prepared aqueous solution was sonicated, and stirred at 800 rpm. A squalene solution mixture, in which squalene was dissolved in a small amount of tetrahydrofuran (THF), was added batchwise to the aqueous solution while stirring the aqueous solution. The solution mixture containing squalene was sonicated for 30 minutes, and stirred at 300 rpm to 400 rpm for 5 hours or more to remove the THF solvent therefrom. Subsequently, a PLL-b-Phe emulsion in an oil-in-water state, in which squalene was carried, was prepared. FIG. 2 illustrates a photograph of the prepared emulsion observed with a TEM microscope. FIGS. 3 and 4 illustrate diagrammatic views of the emulsion in an oil-in-water state and the method of preparing the emulsion.

[0069] 3) Preparation of Vaccine Composition

[0070] 25 l of the emulsion in an oil-in-water state prepared in step 2) was mixed with 25 l of a PBS solution including an influenza virus antigen in a volumetric ratio of about 1:1 such that an HA titer became about 128, thereby preparing a vaccine composition.

[Experimental Example 1] NMR Analysis

[0071] A polymerization result of the amphiphilic poly-amino acid polymer (PLL-b-Phe) was investigated through NMR analysis. Results are shown in FIGS. 5 and 6. FIG. 7 illustrates a diagrammatic view of a polymerization process of PLL-b-Phe. First, H-Lys(Z)OH was reacted with triphosgene to synthesize a Lys(z)-NCA lysine anhydride. NCA and Cbz peaks of the synthesized Lys(z)-NCA lysine anhydride were observed through NMR analysis to confirm whether it was suitably synthesized. The synthesized Lys(z)-NCA lysine anhydride was polymerized using hexylamine to synthesize a cationic poly-amino acid polymer (PLL(z), Poly-l-lysine(z)). Successful polymerization of the PLL(z) was confirmed through the disappearance of NCA peaks in NMR data. Subsequently, a Cbz protective group protecting an amine group was de-protected using an HBr/acetic acid solution, and synthesis of polylysine (Poly-l-lysine), as a cationic amino acid polymer, was confirmed through weakening and disappearance of Cbz peaks in NMR analysis data. In addition, a hydrophobic amino acid anhydride (Phe-NCA) was reacted with polylysine so that a phenylalanine anhydride was polymerized in a branch shape to the polylysine. As a result, polymerization was confirmed as being accomplished through NMR analysis data wherein the NCA peaks disappeared and a benzene ring peak of phenylalanine was generated.

[Experimental Example 2] Measurement of Size Distribution of Amphiphilic Polymer Particles in Oil-in-Water-Type Emulsion State by DLS Method

[0072] The amount of amphiphilic poly-amino acid polymer (PLL-b-Phe) was fixed and the amount of squalene was adjusted to measure a size distribution of the amphiphilic polymer particles in an oil-in-water-type emulsion state. Using the emulsion preparation method of step 2) of Example 1, squalene was carried in the polymer. Particle size changes dependent upon the amount of squalene were investigated using a dynamic light scattering (DLS) method. Results are shown in FIGS. 8 to 10 and Table 1 below.

TABLE-US-00001 TABLE 1 D.W PLL-b-Phe Size (mL) (mg) Squalene RPM (nm, AVG) STDEV {circle around (1)} 20 60 .sup.1%/200 l 900 221.03 1.87 {circle around (2)} 20 60 5%/1 mL 900 255.11 4.25 {circle around (3)} 20 60 10%/2 mL 900 354.47 11.77

[0073] As shown in FIGS. 8 to 10 and the Table 1, it was confirmed that the sizes of the amphiphilic polymer particles in an oil-in-water emulsion state were gradually increased with increasing squalene content. In addition, it was confirmed that the particles were evenly monodispersed through the DLS analysis.

[Experimental Example 3] Measurement of Antibody Titer

[0074] Mice were intramuscularly inoculated at 2-week intervals and, four weeks after the first inoculation, were subjected to an erythrocyte coagulation inhibition experiment to investigate antibody titers. The antibody titers were measured using a hemagglutination inhibition (HI) test method. Experiments were carried out under five conditions: (i) PBS (non-vac of FIG. 11), ii) an influenza vaccine antigen (Ag-only of FIG. 11), iii) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant, AddaVax, for research (AddaVax of FIG. 11), iv) a combination of an influenza vaccine antigen and PLL-b-Phe (Amino-Sq of FIG. 11), v) a combination of an influenza vaccine antigen and an addavax produced in a laboratory (T80-Sq of FIG. 11). In each of the conditions, five mice were used as subjects, and a CA04 (H1N1) virus was used as an immune vaccine antigen. Results are shown in FIG. 11.

[0075] As shown in FIG. 11, it was confirmed that, when an antibody titer was confirmed as an average HI titer value, an antibody titer of the combination of the influenza vaccine antigen and PLL-b-Phe was 1.5 times higher than that of the combination of the influenza vaccine antigen and addavax.

[Experimental Example 4] Confirmation of Stability of Particles

[0076] A storage temperature-dependent particle size of the emulsion including the amphiphilic poly-amino acid polymer (PLL-b-Phe), in which squalene was carried, prepared in step 2) of Example 1 was measured by means of a DLS instrument to investigate the stability of the particles. To investigate storage period-dependent stability of the particles, the particles were stored in a 4 C. refrigerator and at room temperature (RT, 25 C.).

[0077] As shown in FIG. 12, it was confirmed that the initial particle size was maintained even if a storage period was prolonged. From this result, it can be confirmed that the amphiphilic amino acid polymer particles according to the present invention are uniformly dispersed even when stored for a long time.

[Experimental Example 5] Measurement of Immunological Activity in Virus-Infected Models

[0078] 1) Measurement of Immunological Activity in Influenza Virus (H1N1)-Infected Mouse Models

[0079] Mice were intramuscularly inoculated twice at 2-week intervals. Two weeks after the last inoculation, blood samples were collected and subjected to immunological activity measurement experiments using ELISA to investigate an antigen-specific antibody-based immune response (humoral immunity). To measure immunological activity of the mice, each of the collected serum samples was diluted 1:50, and the amounts of IgG and isotypes thereof (IgG1, IgG2a) therein were investigated using enzyme-linked immunosorbent assay (ELISA). Results are shown in FIGS. 13 to 15. Experiments were conducted under six conditions: (i) an untreated group (N.C.: administration of PBS 50 l), ii) an influenza vaccine antigen (Ag: administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+PBS 25 l), iii) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant, Alum (ThermoFisher Scientific, Imject, US) (Ag/Alum: administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+Alum 25 l), iv) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant, AddaVax (InvivoGen, US) (Ag/AddaVax: administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+AddaVax 25 l), v) a combination of an influenza vaccine antigen and squalene (0%)-carrying PLL-b-Phe (Ag/CASq (0%): administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+CASq (0%) 25 l), and vi) a combination of an influenza vaccine antigen and squalene (5%)-carrying PLL-b-Phe (Ag/CASq (5%): administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+CASq (5%) 25 l). Here, the antigen and the immunoadjuvant were mixed in a ratio of 1:1 to prepare a vaccine formulation. For each of the conditions, three mice were subjected to the experiment, and a CA04 (influenza virus H1N1) virus was used as an immune vaccine antigen. Results are shown in FIGS. 13 to 15.

[0080] As shown in FIGS. 13 to 15, it was confirmed that, as results of ELISA experiments conducted to indirectly investigate the amount of IgG at absorbance (450 nm), CASq (5%) showed a higher amount of IgG than AddaVax and Alum through a difference in absorbance intensity. In addition, it was confirmed that, from results for IgG2a related to the Th1 immune response and IgG1 related to the Th2 immune response, CASq (5%) exhibited significantly higher levels.

[0081] 2) Measurement of Infection Inhibition in Influenza Virus (H1N1)-Infected Mouse Models

[0082] Mice were intramuscularly inoculated at 2-week intervals, and then an influenza virus (H1N1) was nasally challenge-inoculated thereto in an content of 20*LD.sub.50 at a dose of 30 ml to investigate an infection inhibition ability of the vaccine according to the present invention. Results are shown in FIG. 16. To investigate the infection inhibition ability, the weights of the animals were measured for 11 days after the virus infection. Here, 10 mice were employed for each of the following six conditions: (i) an untreated group (N.C. of FIG. 16), ii) a PBS-treated group (PBS of FIG. 16: challenge-inoculation after administration of PBS 50 l), iii) an influenza vaccine antigen (Ag of FIG. 16: challenge-inoculation after administration of antigen (inactivated immune antigen, antigen titer: 32) 25 l+PBS 25 l), iv) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant Alum (ThermoFisher Scientific, Imject, US) (Ag/Alum of FIG. 16: challenge-inoculation after administration of an antigen (inactivated immune antigen, antigen titer 32) 25 l+Alum 25 l), v) a combination of an influenza vaccine antigen and a commercially available immunoadjuvant AddaVax (InvivoGen, US) (Ag/AddaVax of FIG. 1: challenge-inoculation after administration of an antigen (inactivated immune antigen, antigen titer 32) 25 l+AddaVax 25 l), and vi) a combination of an influenza vaccine antigen and squalene (5%)-carrying PLL-b-Phe (Ag/CASq (5%) of FIG. 16: challenge-inoculation after administration of an antigen (inactivated immune antigen, antigen titer: 32) 25 l+CASq (5%) 25 l). Here, the antigen and the immunoadjuvant were mixed in a ratio of 1:1 to prepare vaccine formulations.

[0083] In addition, on days 5 and 7 of infection, the lungs of the mice were harvested to measure the titer of the virus remaining in the lungs. Here, three mice were investigated for each of the conditions. Results are shown in FIG. 17.

[0084] As shown in FIG. 17, it was confirmed that a weight change of the experimental group (Ag/CASq(5%)), in which the immunoadjuvant of the present invention was used, was the closest to that of the untreated control group, and the experimental group (Ag/CASq(5%)) exhibited a superior immunosuppressive ability compared to the commercially available immunoadjuvant. In addition, it was confirmed that, from results of the viral titers investigated on days 5 and 7 of infection, the lowest virus titer was exhibited upon use of the immunoadjuvant of the present invention.

[0085] 3) Measurement of Immunological Activity in Porcine Epidemic Diarrhea (PED) Virus-Infected Guinea Pig Models

[0086] Guinea pigs were intramuscularly inoculated three times at 2-week intervals. At two weeks after the second and third inoculations, blood samples were collected from the guinea pigs. The collected blood samples were subjected to immunological activity measurement experiments using ELISA to investigate an antigen-specific antibody-based immune response (humoral immunity). To measure immunological activity in the guinea pigs, the collected serum was diluted 1:50, and the amount of IgG was investigated using enzyme-linked immunosorbent assay (ELISA). Experiments were conducted under the following five conditions: (i) an untreated group (N.C. of FIG. 18: administration of PBS 50 l), ii) a PED immune vaccine antigen (Ag only of FIG. 18: administration of antigen 25 l+PBS 25 l), iii) a combination of PEDK (PED virus immune antigen for muscle inoculation) and a commercially available immunoadjuvant, AddaVax (InvivoGen, US) (Ag/AddaVax of FIG. 18: administration of antigen 25 l+AddaVax 25 l), iv) a combination of PEDK (PED virus immune antigen for muscle inoculation) and a commercially available immunoadjuvant, IMS1313 (SEPPIC, France) (Ag/IMS1313 of FIG. 18: administration of antigen 25 l+IMS1313 25 l), and v) a combination of PEDK (PED virus immune antigen for muscle inoculation) and squalene (5%)-carrying PLL-b-Phe (Ag/CASq(5%) of FIG. 18: administration of antigen 25 l+CASq (5%) 25 l). Here, the antigen and the immunoadjuvant were mixed in a ratio of 1:1 to prepare vaccine formulations. For each of the conditions, two mice were used as subjects. Results are shown in FIG. 18.

[0087] As shown in FIG. 18, it was confirmed that, when the immunoadjuvant according to the present invention was used, a remarkable IgG value was exhibited, compared to the case where a conventional immunoadjuvant used in a vaccine product against the porcine epidemic diarrhea (PED) virus was used. It was confirmed that, from the result of the immunological activity against the PED virus of the immunoadjuvant according to the present invention, the immunoadjuvant according to the present invention was an excellent immunoadjuvant against the PED virus.