KR102355774B1 - An adjuvant composition comprising glycol chitosan derivatives - Google Patents

Abstract

The present invention relates to a glycol chitosan derivative modified with methyl acrylate, a glycol chitosan derivative substituted with a carboxyl group, and an adjuvant composition comprising the same. The adjuvant composition of the present invention and the vaccine composition comprising the antigen exhibited increased immunogenicity compared to a vaccine composition comprising a commercially available adjuvant.

Description

An adjuvant composition comprising glycol chitosan derivatives

The present invention relates to novel adjuvant compositions. More specifically, the present invention relates to a vaccine adjuvant composition based on glycol chitosan.

When a therapeutic vaccine is administered to a patient in order to treat various diseases, the efficiency itself is important, but it is necessary to overcome various internal environments that reduce the efficacy of the vaccine. To this end, a technology for delivering vaccines together using emulsions, liposomes, nanoparticles, etc. has been developed, and recently, adjuvants for immune stimulation, and receptors have been developed. Agents used are being developed (Seth A, Ritchie FK, Wibowo N, Lua LHL, Middelberg APJ. Non-Carrier Nanoparticles Adjuvant Modular Protein Vaccine in a Particle-Dependent Manner. PloS one. 2015;10). In particular, as an adjuvant or an adjuvant as an adjuvant, it can be prepared in a variety of ways, and thus is widely used in the manufacture of vaccines. An ideal adjuvant should have a reproducible manufacturing process and should be non-toxic. As such, *?**?*adjuvant*?**?* refers to an agent that does not constitute a specific antigen, but enhances the strength and lifespan of the immune response to the co-administered antigen, and is safe for immunotherapy. and research for the development of new adjuvants or vaccine adjuvants is continuously being conducted (Batista-Duharte A, Lindblad EB, Oviedo-Orta E. Progress in understanding adjuvant immunotoxicity mechanisms. Toxicology letters. 2011;203:97-105).

Chitosan is a long-chain polymer named N-acetylglucosamine, which is non-toxic, biocompatible, and well-known as a substance derived from nature (Zaharoff DA, Rogers CJ, Hance KW, Schlom J, Greiner JW. Chitosan solution enhances the immunoadjuvant properties of GM-CSF. Vaccine. 2007;25:8673-86). Chitosan derivatives are currently being widely used as gene and drug carriers, and are also being applied in the pharmaceutical field (Haryono A, Salsabila K, Restu WK, Harmami SB, Safari D. Effect of Chitosan and Liposome Nanoparticles as Adjuvant Codelivery on the Immunoglobulin G Subclass Distribution in a Mouse Model. J Immunol Res. 2017; Saenz L, Neira-Carrillo A, Paredes R, Cortes M, Bucarey S, Arias JL. Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine. Pharmaceutics.2009;369:64-71). However, chitosan does not dissolve well in water and only dissolves when an acidic solution is used. On the other hand, glycol chitosan, a derivative of chitosan, is non-toxic, has biocompatibility and biodegradability, and is water-soluble (Pawar D, Mangal S, Goswami R, Jaganathan KS. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV. 2013;85:550-9). It has been reported that glycol chitosan shows a great effect in laser immunotherapy (Chen WR, Liu H, Ritchey JW, Bartels KE, Lucroy MD, Nordquist RE. Effect of different components of laser immunotherapy in treatment of metastatic tumors in rats. Cancer Res. 2002;62:4295-9), which shows that glycol chitosan can be used as a potentially useful adjuvant. In addition, it has been confirmed that the immunostimulatory activity of polyphosphazene-polylactic acid depends on the content of carboxylic acid. It has been shown that increased immunostimulatory activity increases (Andrianov AK, Svirkin YY, LeGolvan MP. Synthesis and biologically relevant properties of polyphosphazene polyacids. Biomacromolecules. 2004;5:1999-2006). Meanwhile, Korean Patent Application No. 10-1998-0703215 describes an influenza vaccine composition comprising chitosan as an adjuvant. Based on these previous studies, it was confirmed that glycol chitosan, a chitosan derivative to be used in the present invention, is water-soluble and has excellent biocompatibility and biodegradability.

Accordingly, the present inventors tried to develop a vaccine adjuvant using a chitosan derivative. Glycol chitosan was used as an adjuvant and this was modified with methyl acrylate. In addition, the end of GC-MA was substituted with carboxylic acid using an ester hydrolysis method. As a result, the present invention has completed the present invention that the glycol chitosan derivative has an excellent effect as an adjuvant or vaccine adjuvant.

Korean Patent Application No. 10-1998-0703215

Seth A, Ritchie FK, Wibowo N, Lua LHL, Middelberg APJ. Non-Carrier Nanoparticles Adjuvant Modular Protein Vaccine in a Particle-Dependent Manner. PloS one. 2015;10. Batista-Duharte A, Lindblad EB, Oviedo-Orta E. Progress in understanding adjuvant immunotoxicity mechanisms. Toxicology letters. 2011;203:97-105. Haryono A, Salsabila K, Restu WK, Harmami SB, Safari D. Effect of Chitosan and Liposome Nanoparticles as Adjuvant Codelivery on the Immunoglobulin G Subclass Distribution in a Mouse Model. J Immunol Res. 2017. Saenz L, Neira-Carrillo A, Paredes R, Cortes M, Bucarey S, Arias JL. Chitosan formulations improve the immunogenicity of a GnRH-I peptide-based vaccine. International journal of Pharmaceutics. 2009;369:64-71. Pawar D, Mangal S, Goswami R, Jaganathan KS. Development and characterization of surface modified PLGA nanoparticles for nasal vaccine delivery: effect of mucoadhesive coating on antigen uptake and immune adjuvant activity. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV. 2013;85:550-9. Chen WR, Liu H, Ritchey JW, Bartels KE, Lucroy MD, Nordquist RE. Effect of different components of laser immunotherapy in treatment of metastatic tumors in rats. Cancer Res. 2002;62:4295-9. Andrianov AK, Svirkin YY, LeGolvan MP. Synthesis and biologically relevant properties of polyphosphazene polyacids. Biomacromolecules. 2004;5:1999-2006.

As such, an object of the present invention is to provide a glycol chitosan derivative that can be used in the preparation of an adjuvant composition.

Another object of the present invention is to provide an adjuvant composition comprising a glycol chitosan derivative having a carboxyl group by hydrolyzing methyl acrylate.

Another object of the present invention is to provide a vaccine composition comprising the adjuvant composition and antigen of the present invention.

Another object of the present invention is to provide a method for immunizing a human or animal comprising administering the vaccine composition to the human or animal in need of administration of the vaccine.

In order to achieve the above object, the present invention provides a methyl acrylate-modified glycol chitosan derivative having the structural formula of the following formula (1):

Formula 1

(Wherein, n is an integer of 1 to 5,000 or less, R ₁ and R ₂ are each independently hydrogen, nitro, C _{1 to C 5} alkyl group, C _{5 to C 10} aryl group, C _{4 to C 10} heteroaryl group, a C ₃ -C ₁₀ cycloalkyl group or a C ₃ -C ₁₀ heterocycloalkyl group).

The present invention also provides an adjuvant composition comprising a methyl acrylate-modified glycol chitosan having the structural formula of Formula 1 above.

Preferably, and as a non-limiting example, the molecular weight of the glycol chitosan derivative is 1,000 Da to 1,000,000 Da, and the glycol chitosan derivative has a methyl acrylate substitution rate of 10% to 100%.

In addition, the present invention provides a vaccine composition comprising the adjuvant composition and the antigen. In the present invention, the antigen is, for example, a bacterial antigen, a viral antigen, a recombinant antigen, or a combination thereof, but is not limited thereto. In the present invention, the bacterial antigen is, for example, Mycoplasma hyopneumoniae , Mycoplasma galanacieum , Mycoplasma bovis , Haemophilus parasuis , Haemophilus somnus , Mycobaterium bovis , Bordetella bronchiseptica , Leptospira , Actinobacillus pleuropneumoniae , Pasteuella multocida , Erysipelothrix rhusiopathiae, Escherichia coli , Streptococcus suis , Streptococcus uberis , Staphylococcus aureus , and Salmonella entericaserovars may be one or more antigens selected from the group consisting of. In the present invention, the viral antigen is, for example, Bovine herpesviruses-1,3,6, Bovine viral diarrhea virus (BVDV) type 1,2, Bovine parainfluenza virus, Bovine respiratory syncytial virus, Bovine leukosis virus, Porcine epidemic diarrhea virus , Porcine reproductive and respiratory syndrome virus (PRRSV), Foot and mouth disease virus, Swine fever virus, Porcine parvovirus, Porcine circovirus, Swine influenza virus, Swine vesicular disease virus, Techen fever virus, Rabies virus, Canine coronavirus, Canine influenzavirus, Canine It may be at least one antigen selected from the group consisting of parvovirus, Feline panleukopenia virus, Feline calicivirus, Feline parvovirus, and Feline herpesvirus. The additional antigen may include, but is not limited to, one or more antigens selected from the group consisting of inactivated whole or partial cell preparations, or in the form of antigen molecules obtained by conventional protein purification, genetic engineering techniques and chemical synthesis. it is not

In addition, the present invention provides a method for preparing a methyl acrylate-modified glycol chitosan derivative. In an embodiment of the present invention, glycol chitosan (GC) and methyl acrylate (MA) are reacted to prepare a methyl acrylate-modified glycol chitosan derivative, and then ester hydrolyzed to methyl acrylate-modified carboxyl group A glycol chitosan derivative having a was prepared. Glycol chitosan and methyl acrylate are reacted, for example, in a molar ratio of 1:50 to 1:200, and ester hydrolysis may be performed using, for example, NaOH, but is not limited thereto.

The adjuvant composition comprising a glycol chitosan derivative having a carboxyl group by hydrolyzing methyl acrylate of the present invention enhances the immune function of bacteria, viruses, and/or recombinant antigens. In one embodiment of the present invention, as a result of comparing cell viability in two cell lines using glycol chitosan derivative, which is the adjuvant composition of the present invention, and commercially used alum gel, etc. as a control, the sample using the composition of the present invention was It showed a high survival rate of over 90%. From this, it was confirmed that the glycol chitosan derivative prepared in the present invention does not cause an immune response and does not exhibit cytotoxicity when delivered into cells, and it is an adjuvant composition with excellent safety when injected into a living body. In another embodiment, the survival rate of mice administered with a vaccine composition comprising a glycol chitosan derivative of the present invention together with a bacterial antigen increased. In addition, in another embodiment, a significant increase in antibody titer was observed in guinea pigs administered with a vaccine composition comprising glycol chitosan of the present invention together with a viral antigen. Therefore, the glycol chitosan of the present invention may be effectively used as an adjuvant in the preparation of a vaccine composition.

Figure 1 (A) schematically shows the synthesis process of glycol chitosan methyl acrylate, (B) is a base-catalyzed ester hydrolysis process of ^{glycol chitosan methyl acrylate COOH or COO -} Na ^{+ schematically shows.}
Figure 2 shows the 1H NMR spectrum of glycol chitosan (GC, black line) (top), (B) glycol chitosan methyl acrylate (bottom), respectively.
3 is an ATR-FTIR spectrum of glycol chitosan (GC, black line), glycol chitosan methyl acrylate (GC-MA, green line) and glycol chitosan methyl acrylate COOH/COO ^- Na ⁺ (GC-MA-COOH/ COO ^- Na ⁺ , red line) respectively.
4 shows the results of evaluation of adjuvant efficacy using the recombinant protein antigen.
5 shows the results of evaluation of adjuvant efficacy using bacterial antigens.
6 shows the results of evaluation of adjuvant efficacy using a viral antigen.
7 shows the results of toxicity evaluation using the HEK 293 cell line. Alum (Alum, blue line), glycol chitosan (GC, red line), glycol chitosan methyl acrylate (GC-MA, green line) and glycol chitosan methyl acrylate COOH/COO ^- Na ⁺ (GC-MA-COOH/COO ^- Na ⁺ , purple line) respectively.
8 shows the results of toxicity evaluation using the HeLa cell line, and each line is the same as in FIG. 7 .

The present invention will be described in more detail below based on examples and drawings. The following examples and accompanying drawings are provided to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Materials and Methods

Glycol chitosan (GC, degree of deacetylation = 91.6%) was purchased from Wako pure chemical industrials, Osaka, Japan. Methyl acrylate (99%), sodium hydroxide, and methanol (anhydrous, 99.8%) were purchased from Sigma-aldrich. Dialysis membrane tubing was purchased from Spectrum Labs, USA.

(1) Synthesis of glycol chitosan methyl acrylate (GC-MA)

30 mg of glycol chitosan (GC, 1 mmol) and 1.305 ml of methyl acrylate (MA, 100 mmol) were weighed. The measured glycol chitosan was dissolved in tertiary distilled water and transferred to a tube. The prepared 250 ml round bottom flask and magnetic bar were washed with methanol. Methyl acrylate was placed in a round-bottom flask, filled with methanol, and then, while stirring for about 10 minutes, glycol chitosan pre-dissolved in distilled water was dropped. The solvent in the flask was adjusted in a ratio of methanol and distilled water to 9:1. After filling the inside of the round flask with nitrogen gas, it was reacted at 37° C. and 180 rpm for 4 days in a shaking incubator. After 4 days, methanol was removed by evaporation, and the remaining product was placed on a dialysis membrane (MWCO 3.5 kDa) and then dialyzed against distilled water for 1 day. After 1 day, the product was freeze-dried for 1 day again to remove water. ^{At this time, 1} H Nuclear Magnetic Resonance spectroscopy (AVANCE, 600 FT-NMR, Bruker, Germany) was measured to confirm the binding of GC-MA. GC: ¹ H-NMR (600 MHz, D ₂ O, ppm) δ 2.0 - 2.1 (NHCOC H ₃ , acetyl group), 2.6 - 2.8 (H2 of deacetylated monomer), 3.4 - 3.9 (H2 to H8, multiplet, D -glucosamine unit and hydroxyl ethyl substituents), 4.3-4.5 (H1); GC-MA: ¹ H-NMR (600 MHz, D ₂ O, ppm) δ 2.0 - 2.1 (NHCOC H ₃ , acetyl group), 2.5-2.7 (H2 of deacetylated monomer, OCC H , H10), 3.0 - 3.2 ( HCC H N, H9), 3.5 - 3.8 (H2 to H8, multiplet, D-glucosamine unit and hydroxyl ethyl substituents), 4.4-4.5 (H1)

(2) GC-MA-COOH production

The synthesized GC-MA was dissolved in 5M sodium hydroxide (NaOH) and then transferred to a 250 ㎖ round flask. After adjusting the temperature of the water to 70 ~ 80 ℃, the reaction was carried out for 3 hours while stirring the round flask with a bath. After 3 hours, the GC-MA dissolved in the round flask was put into a dialysis membrane (MWCO 3.5 kDa) and dialyzed in distilled water for 1 day. After 1 day, the product was lyophilized to remove water. The functional group of the synthesized GC-MA-COOH was confirmed (Nicolet iS5 Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) spectroscopy (Thermo scientific, Madison, Wisconsin, USA).

(3) Confirmation of GC-MA-COOH synthesis

Glycol chitosan was used as a backbone and methyl acrylate was connected by a Michael addition method. The synthesis process is shown in FIG. 1(A), and the process of GC-MA-COOH prepared using NaOH after GC-MA synthesis is shown in FIG. 1(B). Next, in order to confirm that GC and MA were combined, the synthesized material _{was dissolved in D 2} O, and then ^{measured by 600 MHz 1} H NMR nuclear magnetic resonance spectrum. As a result, as shown in FIG. 2, when MA was connected to GC, it was confirmed that hydrogen (H10) of OCC H- was present between 2.5 and 2.7 ppm, and hydrogen of HCC H N- (H9) at 3.0 - 3.2 ppm. ), it was confirmed that the synthesis of GC-MA was well accomplished. GC-MA-COOH obtained by ester hydrolysis of the synthesized GC-MA with 5 M NaOH was confirmed through the ATR-FTIR spectrum to the functional group generated after the synthesis. As a result, as shown in FIG. 3 , 3500 cm ⁻¹ in the graph of GC is a hydroxyl group, and when MA is bound to GC, it can be seen that a carboxyl group appears at ^{1550 cm −1 .} It could be confirmed that _{NCH 2} CH ₂ COO CH ₃ - was formed through the Michael addition method of GC and MA. In particular, when GC-MA was hydrolyzed with 5M NaOH ^{, new peaks appeared at 1420 and 1610 cm -1} . This peak is a salt form of COOH when _{5M NaOH meets OOCH 2 at the} end of GC-MA and removes water. It was found that Na ⁺ was combined in the .

(4) Toxicity evaluation of GC-MA-COOH

To confirm the intracellular toxicity of the synthesized GC-MA-COOH, toxicity evaluation was performed using HeLa and HEK 293 cell lines. At this time, the cell viability was shown in FIGS. 7 and 8 by measuring each absorbance after treatment with the WST reagent. For comparison with GC-MA-COOH, alum gel, glycol chitosan (GC) and non-hydrolyzed glycol chitosan methyl acrylate (GC-MA) were used as controls. The concentrations of the samples used were 12.5, 25, 50, 100 and 200 ug/ml to the cells using concentrations. As a result, FIG. 7 shows that the HEK 293 cell line showed a survival rate of 90% or more in all samples. 8 is a HeLa cell line, and the cell viability was 90% or more as in FIG. 7 . This is a result that proves that glycol chitosan methyl acrylate COOH synthesized based on glycol chitosan is biocompatible as it is nontoxic, harmless, and has high safety as an adjuvant.

Example

Evaluation of adjuvant efficacy using recombinant protein antigen

Efficacy evaluation using recombinant protein antigen was performed. First, five types of test vaccines were prepared with the composition shown in Table 1 below. The glycol chitosan polymer (GC-MA-COOH) prepared in Preparation Example was used.

test vaccine Vac1 Vac2 Vac3 Vac4 Vac5 Antigen PPV VP2
HA 2 ¹¹ PPV VP2
HA 2 ¹¹ PPV VP2
HA 2 ¹¹ PPV VP2
HA 2 ¹¹ - Polymer GC
0.1 mg/dose GC-MA
0.1 mg/dose GC-MA-COOH
0.1 mg/dose - - note Non adjuvant unvaccinated group

Porcine parvovirus VP2 protein (Recombinant Baculovirus VP2-BV strain) (seed obtained from: National Veterinary Science and Quarantine Service) was used as an antigen, and HA 2 ¹¹ /ml was included in the vaccine composition. All test vaccines were adjusted to have a pH of 7. As an experimental animal, a guinea pig (male, 300-350 g) was used. Five guinea pigs per test vaccine group (3 in the control group) were subcutaneously inoculated by 1/2 (1 ml/two) of the prepared vaccine, and after 4 weeks, blood was collected together with the control group to measure the hemagglutination inhibitory antibody titer. . Table 2 and Figure 4 below show the results.

vaccine group Vac1 Vac2 Vac3 Vac4 Vac5 Sample GC GC-MA GC-MA-COOH Non adjuvant unvaccinated group inoculation group 320 160 640 80 One 160 160 640 80 One 320 160 320 160 One 160 80 320 40 - 160 80 80 80 - geometric mean 211.1 121.3 320 80 One Standard Deviation 87.6 43.8 240 43.8 0

As shown in Table 2 and FIG. 4, according to the results of the hemagglutination inhibitory antibody, Vac3 (GC-MA-COOH) of the present invention exhibited the highest hemagglutination inhibitory antibody titer, and glycol chitosan polymer ( GC-MA-COOH) precursors (Vac1, Vac2) also exhibited higher hemagglutination inhibitory antibody titers compared to the non-adjuvant and unvaccinated groups, which is due to the adjuvant effect. .

Evaluation of adjuvant efficacy using bacterial antigens

Efficacy evaluation using bacterial antigens was performed. First, four types of test vaccines were prepared with the composition shown in Table 3 below. The glycol chitosan polymer (GC-MA-COOH) prepared in Preparation Example was used.

test vaccine Vac1 Vac2 Vac3 Vac4 Antigen SE bacterin
0.2 X 10 ¹⁰ CFU/ml SE bacterin
0.2 X 10 ¹⁰ CFU/ml SE bacterin
0.2 X 10 ¹⁰ CFU/ml - Polymer GC-MA-COOH
0.4 mg/dose - - - Alum - Alum 20% - - note positive control Non adjuvant unvaccinated group

SE bacterin ( Erysipelothrix . rhusiopathiae ) (Seed source: National Veterinary Science and Quarantine Service, antigen source: Central Vaccine Research Institute) was used as an antigen (antigen), ^{and 0.2 X 10 10} CFU/ml was included in the vaccine composition. As a positive control, alum (aluminum hydroxide gel), a commonly used vaccine adjuvant, was used, and 20% (v/v) was included in the vaccine composition. All test vaccines were adjusted to have a pH of 7. Eight mice (ICR mice, males, 4 to 6 weeks old, 15 to 20 g) per test vaccine group were inoculated with 1/10 (0.2 ml) of the prepared vaccine once subcutaneously, and 5 mice were treated as a control group. (uninoculated group). Pig single bacteria ( E. rhusiopathiae ) together with the control group 2 weeks after the first inoculation After subcutaneous challenge inoculation with 100 LD ₅₀ /0.2ml, survival was observed for 10 days. Tables 4 and 5 below show the results.

vaccine group Sample Marisu Test result Survival rate (%) Vac1 GC-MA-COOH 10 8/10 80 Vac2 Alum 10 7/10 70 Vac3 Non adjuvant 10 6/10 60 Vac4 unvaccinated group 5 0/5 0

As confirmed in Table 4 and FIG. 5, according to the survival rate results, the positive control group Vac2 (Alum) and the Vac1 (GC-MA-COOH) of the present invention were more effective than those of Vac4 (uninoculated group). It showed a high survival rate, which is due to the adjuvant effect. In particular, Vac1 (GC-MA-COOH) of the present invention showed more excellent effects than Vac2 (Alum).

Evaluation of adjuvant efficacy using viral antigens

Efficacy evaluations using viral antigens were performed. First, four types of test vaccines were prepared with the composition shown in Table 5 below.

group Vac1 Vac2 Vac3 Vac4 Antigen CCV
10 ^5.5 TCID ₅₀ /dose CCV
10 ^5.5 TCID ₅₀ /dose CCV
10 ^5.5 TCID ₅₀ /dose - Polymer GC-MA-COOH
0.4mg/dose - - - Alum - Alum 3% - - note - positive control Non adjuvant unvaccinated group

As an antigen (antigen), canine corona virus (CCV, Canine Corona virus) (seed: Cornell university, or antigen: Central Vaccine Research Institute) was used, and 10 ^5.5 TCID ₅₀ /ml was included in the vaccine composition. As a positive control, aluminum hydroxide gel, a commonly used vaccine adjuvant, was used, and 3% (v/v) was included in the vaccine composition. All test vaccines were adjusted to have a pH of 7. A guinea pig (male, 300 ~ 350g) was used as an experimental animal. Five guinea pigs per test vaccine group (3 in control group) were intramuscularly inoculated twice (1 ml/two) of the prepared vaccine twice at an interval of 2 weeks, and after 2 weeks, blood was collected with the control group and canine corona Neutralizing antibody against virus (NPLA) was measured. Table 6 and Figure 6 below show the results.

vaccine group Vac1 Vac2 Vac3 Vac4 Sample GC-MA-COOH Alum Non adjuvant unvaccinated group inoculation group 32 32 One One 16 2 One One 32 16 2 One 32 16 One - 16 8 2 - geometric mean 24.25 10.56 1.32 One Standard Deviation 8.76 11.27 0.54 0

According to the national examination standard for veterinary medicines for canine coronavirus, 80% or more of inoculated animals must satisfy 8 times or more. As a result of the neutralizing antibody test, it was confirmed that the antibody titer of the Vac1 (GC-MA-COOH) group of the present invention was higher than that of Vac2 (secondary). As a result of the neutralizing antibody test, it was confirmed that the antibody titer of the Vac1 (GC-MA-COOH) group was formed the highest. Despite the fact that the test vaccine was prepared by reducing the antigen content of the existing CCV vaccine by half, all inoculated animals of the Vac1 (GC-MA-COOH) group showed an antibody titer more than 8 times the national standard for veterinary drugs. These results suggest that the adjuvant (GC-MA-COOH) of the present invention has a very good effect compared to the currently used alum vaccine adjuvant.

As a result of administering the vaccine composition comprising the adjuvant composition and the antigen according to the present invention to animals, it was confirmed that increased viability and antibody titer were exhibited. Accordingly, the present invention can be widely used for enhancing immunogenicity in the preparation of vaccine compositions for humans or animals.

Claims (16)

A methyl acrylate-modified glycol chitosan derivative having the structural formula of Formula 1 below:
Formula 1

(Wherein, n is an integer of 1 to 5,000 or less, and R ₁ and R ₂ are each independently hydrogen or a sodium salt).
delete As an adjuvant composition comprising the glycol chitosan derivative of claim 1, the molecular weight of the glycol chitosan derivative is 1,000 Da to 1,000,000 Da, the adjuvant composition. The adjuvant composition according to claim 3, wherein the glycol chitosan derivative has a methyl acrylate substitution rate of 10% to 100%. A vaccine composition comprising the glycol chitosan derivative of claim 1 or the adjuvant composition of claim 3 and an antigen. A vaccine composition comprising the adjuvant composition of claim 4 and an antigen. 6. The vaccine composition of claim 5, wherein the antigen is a bacterial antigen, a viral antigen, or a recombinant antigen. 7. The vaccine composition of claim 6, wherein the antigen is a bacterial antigen, a viral antigen, or a recombinant antigen. 6. The method of claim 5, wherein the bacterial antigen is Mycoplasma hyopneumoniae , Mycoplasma galanacieum , Mycoplasma bovis, Haemophilus parasuis , Haemophilus somnus , Mycobaterium bovis , Bordetella bronchiseptica , Leptospira, Actinobacillus pleuropneumoniae , Pasteuella multocida , Erysipelothrix rhusiopathiae , Escherichia coli , Streptococcus suis , Streptococcus uberis , Staphylococcus aureus , and Salmonella entericaserovars . According to claim 5, wherein the viral antigen is Bovine herpesviruses-1,3,6, Bovine viral diarrhea virus (BVDV) type 1,2, Bovine parainfluenza virus, Bovine respiratory syncytial virus, Bovine leukosis virus, Porcine epidemic diarrhea virus, Porcine reproductive and respiratory syndrome virus (PRRSV), Foot and mouth disease virus, Swine fever virus, Porcine parvovirus, Porcine circovirus, Swine influenza virus, Swine vesicular disease virus, Techen fever virus, Rabies virus, Canine coronavirus, Canine influenzavirus, Canine parvovirus, Feline A vaccine composition, wherein the vaccine composition is selected from the group consisting of panleukopenia virus, Feline calicivirus, Feline parvovirus, and Feline herpesvirus. The vaccine composition of claim 9, wherein the bacterial antigen is Erysipelothrix . rhusiopathiae. The vaccine composition of claim 10, wherein the viral antigen is Canine Corona virus. i) reacting glycol chitosan with methyl acrylate by Michael addition to produce methyl acrylate modified glycol chitosan;
ii) ester hydrolysis of the methyl acrylate-modified glycol chitosan obtained in i) to prepare a glycol chitosan derivative having the structure of the following formula (1):
Formula 1

(Wherein, n is an integer of 1 to 5,000 or less, and R ₁ and R ₂ are each independently hydrogen or a sodium salt). The method according to claim 13, wherein in i) glycol chitosan and methyl acrylate are reacted in a molar ratio of 1:50 to 1:200, and the ester hydrolysis of ii) is performed in NaOH, to prepare a glycol chitosan derivative How to. delete delete

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