KR102075573B1 - Adjuvant Compositions Comprising Mastoparan-V1 Peptide as Active Ingredient - Google Patents

Abstract

The present invention relates to an immunoadjuvant composition comprising a mastoparan-V1 peptide as an active ingredient, and more particularly to an immunoadjuvant composition comprising a mastoparan-V1 peptide as an active ingredient, and mastoparan as the immunoadjuvant component. A vaccine composition comprising a -V1 peptide and an antigen.
The mastoparan MP-V1 peptide of the present invention acts as an adjuvant to increase the vaccine effect against pathogenic bacteria and exerts very good immunostimulatory efficacy against certain pathogenic bacteria. Since the vaccine composition of the present invention can economically produce bactericidal vaccines which are safe and have dramatically improved immune induction ability, it is thought that they can substantially help livestock farms.

Description

Adjuvant Compositions Comprising Mastoparan-V1 Peptide as Active Ingredient}

The present invention relates to an immunoadjuvant composition comprising a mastoparan-V1 peptide as an active ingredient, and more particularly to an immunoadjuvant composition comprising a mastoparan-V1 peptide as an active ingredient, and mastoparan as the immunoadjuvant component. A vaccine composition comprising a -V1 peptide and an antigen.

Adjuvant plays an important role in the prevention and treatment of infectious diseases by activating immune cells. In general, an adjuvant is a generic term for substances and methods that regulate (enhances, inhibits) the immune response, and acts on the dosage form of the antigen and acts on immunoreactive cells to augment an immune response in an antigen-specific or non-specific manner. Refers to a substance to inhibit. As a substance with regulatory activity centered on strengthening the immune response, cell components such as BCG and Propionibacterium acnes, cell wall, cell constituents such as trehalose dimycolette (TDM), lipopolysaccharide (LPS) which is endotoxin of gram-negative bacteria B) proteins derived from biological components such as thymic hormones, thymic factors, and taftins, as well as synthetic compounds such as lipid A fraction, β-glucan (polysaccharide), N-acetyl muramyldipeptide (MDP), bestatin, and levamizole; Peptide materials are known.

In addition, lymphokine, monocaine or interferon, cyclophosphamide, which regulates the immune response by removing inhibitory T cells, inhibitory macrophages, and the like, may also be treated as broad immune supporters. . As an adjuvant for the antigen dosage form, Freund's incomplete adjuvant (FIA) formulated with a water-in-oil emulsion containing an equal amount of mineral oil including Alacel A and an aqueous solution of an antigen is used as an immunoadjuvant.

These adjuvants not only enhance the immune response depending on the route of administration, dose, and timing of administration, but also cause the effects of inhibition. Also, depending on the type of adjuvant, the production of blood antibodies against antigen, induction of cellular immunity, and immunoglobulin It is known that there is a difference in class.

On the other hand, researches on the development of dietary supplements, feed and feed additives, and the development of immunostimulants have been actively conducted to improve the animal's self-defense ability against pathogenic bacteria. However, antibiotic-resistant bacteria have arisen due to the administration of an excessive amount of antibiotics for the treatment of animal diseases including humans, and the problem of resistance to antibiotics is becoming increasingly serious. Currently, new antibiotics to replace vancomycin have not yet been developed, and international organizations have been working to try to reduce their resistance by regulating the use of antibiotics.

In addition, the administration of an excess of antibiotics to livestock can threaten the stability as food, which leads to a decrease in commodity, which causes a large economic loss to the livestock farms. In addition, the use of antibiotics and antimicrobials in animal feed mixed with compounded feeds has been banned since 2012. Overseas, the World Health Organization (WHO) warns of the dangers of using antibiotics for livestock feed, and the European Union (EU) has also restricted the use of antibiotics for therapeutic purposes.

As such, there is an urgent need for the explosive increase in bacterial diseases due to the abuse caused by the abuse of antibiotics, the development of methods to prevent animal diseases without the use of antibiotics, and the role of preventive vaccines to increase. It is becoming.

Traditional vaccines against existing pathogenic bacteria are mainly killed vaccines or subunit vaccines. However, conventional vaccines are produced by treatment or heat treatment of chemicals such as formalin, betapropriolactone (BPL) and binary ethyleneimine (BEI), so that extracellular appendages are released or natural epitopes are removed. There is a disadvantage in that the immunogenicity is lowered due to failure to maintain), and a decrease in immuno-induced ability is often caused when the bacterial vaccine is treated to induce an immune response. For example, according to a study by Shuai-Cheng Wu et al., Mice inoculated with formalin-inactivated Salmonella typhimurium (FIST) had a lethal dose (1 × 10 ¹⁰ CFU / mouse, n = 10). Intraperitoneal injection of Salmonella typhimurium showed only a 30% survival rate (Shuai-Cheng Wu, International Immunopharmacology 25, 2015).

In order to compensate for such drawbacks, referring to Patent Registration No. 10-1797276, Patent Publication No. 10-2017-0102095, Patent Registration No. 10-1774863, Patent Publication No. 10-2017-0081569, etc. Attenuated highly virulent strains have been prepared for attenuated vaccines. However, the live attenuated vaccine attenuates pathogenic strains, but because of the use of live bacteria, there is a possibility of causing aversion, and if the gene lost for live attenuation is recovered in the natural state, it may be converted into the original strongly toxic strain. (Lin and He, 2012, Avila-Calderon et al., 2013; Langemann et al., 2010). Therefore, there is a continuous demand for the development of a method for producing a vaccine that is as stable as a bacteriophage vaccine while retaining the ability to induce immunity such as a live attenuated vaccine.

Against this background, as a result of earnest research to develop a new adjuvant to enhance the immune-inducing ability of the low-cost bacterium vaccine produced by the treatment of formalin, which is most commonly used, mastoparan When the MP-V1 peptide is used as an adjuvant, it was confirmed that the immunity of immunity can be dramatically improved to a degree similar to that of the live attenuated vaccine while maintaining the safety of the bacteriostatic vaccine. .

Accordingly, a main object of the present invention is to provide a mastoparan MP-V1 peptide, which serves as an adjuvant that can dramatically improve the immunity of the low-cost bacterium vaccine prepared by formalin and the like. There is.

It is another object of the present invention to provide a method for preparing a vaccine using an adjuvant containing the mastoparan MP-V1 peptide as an active ingredient, and a vaccine according thereto.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides an adjuvant composition comprising a mastoparan MP-V1 peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The inventors have researched and analyzed for many years to find an adjuvant that enhances the vaccine effect of the vaccinated vaccine treated with chemicals such as formalin, which are most commonly used in livestock farming, because they can be manufactured at low prices. As a result, the effect of the mastoparan peptide could be used as an adjuvant was verified. As a result, applying the mastoparan peptide as an adjuvant (adjuvant), it was confirmed that it can significantly increase the immune-inducing ability of the bactericidal vaccine having a safe but low immuno-inducing ability.

In the present invention, the term "mastoparan" means tetratecapeptides contained in bee venom (Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH). ₂ ) and its analogous peptides, in addition to the bee venom belonging to the bee family is a concept that includes all of the peptides of the sequence similar to the peptide sequence. Typically, the mastoparan peptide may be defined collectively as a mastoparan group. The mastoparan is rich in hydrophobic and basic amino acids, taking a random conformation in the hydrophilic environment but having an amphiphilic α-helix structure in the hydrophobic environment.

As used herein, the term "peptide" refers to a polymer consisting of amino acids linked by amide bonds (or peptide bonds). For the purposes of the present invention, it means a peptide having a potentiating activity of the immune response. The peptide structure of the present invention is characterized by having an α-helix structure. In the present invention, the peptide is preferably an antimicrobial peptide having an amino acid sequence of SEQ ID NO: 1. The mastoparan peptide having the amino acid sequence of SEQ ID NO: 1 is a mastoparan peptide isolated by analyzing the whole expression genome and protein of the native expression of Korean native bumblebee. The peptide is a mastoparan (MP-V1) peptide having an amino acid sequence represented by SEQ ID NO: 1 consisting of 15 amino acids of INWKKIKSIIKAAMN.

Examples of the mastoparan peptides having a similar effect to the mastoparan peptide of SEQ ID NO: 1 include mastoparan-L having an amino acid sequence of SEQ ID NO. And mastoparan-B having an amino acid sequence (LKLKSIVSWAKKVL) of Toparan-X (V) and SEQ ID NO: 4.

Compared with other known mastoparans, the mastoparan (MP-V1) peptide of SEQ ID NO: 1 shows a markedly improved immune response enhancing activity compared to other mastoparans, and thus the amino acid of SEQ ID NO: 1 The mastoparan peptide having a sequence has an advantage of acting as an adjuvant more effectively than other mastoparan peptides.

In the present invention, the immunoadjuvant composition is characterized in that the immunoadjuvant composition for a vaccine against pathogenic bacteria. Examples of the pathogenic bacteria which can increase the vaccine effect by acting as an immunoadjuvant of the mastoparan peptide include Salmonella strain, Vibrio strain, Mycobacterium strain, Shigella strain, Escherichia coli, Star Phyllococcus strain, Streptococcus strain, and Listeria strain. More specifically, the strain of genus Salmonella is a genus of Salmonella. In addition, as the type of the pathogenic bacteria Vibrio cholera , mycobacterium bovis , Shigella dysenteriae , Shigella flexneri flexneri ), enteric noxious Escherichia coli, Staphylococcus aureus , Streptococcus suis , Listeria monocytogenesis strains, and the like.

In the adjuvant composition of the present invention, the mastoparan MP-V1 peptide comprises (i) a polynucleotide encoding the mastoparan MP-V1 protein of SEQ ID NO: 1; (Ii) an expression control sequence operably linked with the polynucleotide of (iii) to induce expression of the polynucleotide; And (iii) a recombinant expression vector comprising a transcription termination sequence is produced by the transformed cells into which the recombinant expression vector is introduced.

The contents related to the transformed cells into which the recombinant expression vector to produce the mastopararan MP-V1 peptide of the present invention are introduced are described in detail in Patent Application No. 10-2017-0138257 filed by the present inventors.

In addition, the immunoadjuvant composition of the present invention may exert a sufficient immune response with only the mastoparan MP-V1 peptide to exert prophylactic efficacy against various diseases. Optionally, the immunoadjuvant composition of the present invention may further add other immunoadjuvant components. Group 2 elements selected from the group consisting of Mg, Ca, Sr, Ba and Ra or salts thereof; A Group 4 element selected from the group consisting of Ti, Zr, Hf and Rf; Salts of aluminum or hydrates thereof; Or dimethyloctadecylammonium bromide may be further included. The salts are formed with, for example, oxides, peroxides, hydroxides, carbonates, phosphates, pyrophosphates, hydrogenphosphates, dihydrogenphosphates, sulfates or silicates.

In addition, the immunoadjuvant composition of the present invention additionally contains magnesium hydroxide, magnesium carbonate hydroxide pentahydrate, titadium didoxide, calcium phosphate, calcium carbonate, barium oxide, barium hydroxide, barium peroxide, barium It may further include components such as sulfate, calcium sulfate, calcium pyrophosphate, magnesium carbonate, magnesium oxide, aluminum hydroxide, aluminum phosphate, hydrated aluminum potassium sulfate and the like.

According to another aspect of the invention, the invention provides an antibody comprising (a) an antigen; And (b) provides a vaccine composition comprising a mastoparan MP-V1 peptide having the amino acid sequence of SEQ ID NO: 1 as an adjuvant component as an active ingredient.

As used herein, the term "antigen" is a substance that induces an immune response of a recipient. Therefore, the present invention can be used without limitation any substance exhibiting such an immune response inducing effect.

According to one embodiment of the invention, the antigen may be a peptide, protein, nucleic acid, sugar, pathogen, attenuated pathogen, inactivated pathogen, virus, virus-like particle (VLP), cell or cell fragment. have.

According to one embodiment of the present invention, the antigen is Salmonella antigen, Mycobacterium tuberculosis antigen, Anthrax antigen, Antigen of HAV (Hepatitis A virus), Antigen of Hepatitis B virus (HBV), Antigen of HCV (Hepatitis C virus), HIV (human immunodeficiency virus) antigen, influenza virus antigen, HSV (Herpes simplex virus) antigen, Hib (Haemophilus influenzae type b) antigen, antigen of Neisseria meningitidis, Conifer bacterium diphtheria ( Group consisting of antigens of Corynebacterium diphtheria, antigens of Bordetella pertussis, antigens of Clostridium tetani, antigens of human papilloma virus, and antigens of Varicella virus It can be selected from, and preferably treated with chemicals, such as formalin, betapropriolactone (BPL), binary ethyleneimine (BEI) or inactivated pathogens through heat treatment It may be an antigen made.

The vaccine composition of the present invention can exert a sufficient immune response with only its basic composition, ie, the antigen and the mastoparan MP-V1 peptide, thereby exerting a preventive effect on a specific disease. Optionally, the vaccine composition of the present invention may further comprise the additional adjuvant mentioned above.

According to one embodiment of the invention, the vaccine is Salmonella vaccine, cancer vaccine, flu vaccine, tuberculosis vaccine, anthrax vaccine, HAV vaccine, HBV vaccine, HCV vaccine, HIV vaccine, herpes simplex vaccine, meningitis vaccine, diphtheria vaccine, whooping cough Vaccine, tetanus vaccine or chickenpox vaccine.

The vaccine composition of the present invention can be administered as a vaccine to induce immunity against diseases caused by infection by pathogenic bacteria, wherein the vaccine composition can be suitably formulated with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as oral administration binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors, and in the case of injections, physiological saline And non-aqueous solvents such as aqueous solvents such as ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers and the like can be used in combination. In the case of propellants, suitable propellants, such as compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low boiling point solvents, can be conveniently delivered in the form of aerosol spray presentation from a pressurized pack or sprayer.

The formulation of the vaccine composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elesir, suspensions, syrups, wafers, etc., and in the case of injections, may be prepared in unit dosage ampoules or multiple dosage forms.

The route of administration of the vaccine composition can be administered via any general route as long as it can reach the target tissue.

The term "administration" of the present invention means introducing any substance into a human or animal by any suitable method and is formulated for human or veterinary administration and administered by various routes. The vaccine composition of the present invention may be administered by a parenteral route such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous, and may be administered by inhalation via oral, nasal, rectal, transdermal or aerosol. It may also be administered by bolus or slowly infused, but specifically may be administered by sublingual, intramuscular or nasal route, more specifically nasal route, but is not limited thereto.

Vaccine compositions of the invention comprising an antigen and a mastoparan MP-V1 peptide as an adjuvant component can be administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to exhibit a vaccine effect and an amount not causing side effects or severe or excessive immune responses, and an effective dose level is used to determine the disorder, disorder to be treated. Severity, activity of a particular compound, route of administration, rate of elimination, duration of treatment, drug used in combination or concurrently with the vaccine, age, weight, sex, eating habits, general health status of the subject, and known in the pharmaceutical and medical arts It will depend on various factors including factors. Various general considerations that are considered in determining a “therapeutically effective amount” are known to those skilled in the art, for example, Gilman et al., Eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990. And Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

When administering the vaccine composition of the present invention, the number of dead bacteria as an antigen to be administered in one administration is usually about 1 × 10 ⁷ to 1 × 10 ¹¹ , more specifically 1 × 10 ⁸ to 5 × 10 ¹⁰ , more specifically For example, the range of 5 × 10 ⁸ to 2 × 10 ¹⁰ germs, and the concentration of mastoparan MP-V1 peptide administered as an adjuvant component is 0.1 μg / ml to 100 μg / ml.

The vaccine composition of the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a vaccine composition containing a mastoparan MP-V1 peptide and its active ingredient as an adjuvant component.

(Ii) The mastoparan MP-V1 peptide of the present invention acts as an adjuvant to increase the vaccine effect against pathogenic bacteria and exert very good immunostimulatory efficacy against certain pathogenic bacteria.

(Iii) Accordingly, the vaccine composition of the present invention containing the mastoparan MP-V1 peptide as an adjuvant component can be economically produced for safe and immunization-induced bacteriophage vaccine, so that it can be produced in livestock farms. It can be practically helpful.

1 is a diagram comparing the primary structure and the secondary structure of the mastoparan, the figure comparing the primary structure (Fig. 1A), peptide phylogenetic diagram (Fig. 1B), and circular light dichroism spectrum analysis (Fig. 1C) results.
Figure 2 is a graph measuring the vaccine effect against the highly toxic microorganism Salmonella gallinarum pathogen through the inoculation of the bacteriophage vaccine prepared by adding the mastoparan peptide as an adjuvant component.
Figure 3 is a graph showing the results of the ELISA immune response to IgG protein inoculated with the bacteriophage vaccine prepared by adding the mastoparan peptide as an immunoadjuvant component.
Figure 4 is a graph showing the results of performing ELISA immune response to intramuscular inoculation of the bacteriophage vaccine prepared by adding a mastoparan peptide as an immunoadjuvant component, IFN-γ protein (pg / ml).
Figure 5 is a highly toxic microorganism Salmonella Guerlin arum (Salmonella gallinarum ) Liver of a chicken that survived 2 weeks after challenge with a pathogen, followed by autopsy.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example. Experimental Material Preparation

1-1. Pathogenic Bacteria and Growth Conditions

Salmonella Gallinarum HJL465, a malignant strain isolated from Korean chickens, was selected as a pathogenic bacterium. Pathogenic bacteria Salmonella guerlinum was prepared by incubating in LB broth and LB agar medium (Becton Dickinson, Sparks, MD, USA) at 37 ℃.

1-2. Mastopranan Peptide Synthesis and Purification

The mastoparan peptide is a conventionally loaded link amide resin (100 mg) of 0.61 mmol / g, unless otherwise stated, using 9-fluorenylmethoxycarbonyl (Fmoc, 9-fluorenylmethoxy carbonyl) as a protecting group of amino acids. It was synthesized by solid phase peptide synthesis (SPPS).

The resin was washed with DMF (N, N-dimethylformamide) solution for 45 minutes before synthesis for proper expansion. For sequence extension, the amino acid (5 equivalents) protected with 9-fluorenylmethoxycarbonyl group was HBTU [2- (1H-benzotriazol-1-yl) -1,1 in DMF solution (2 mL, 0.15 mM). , 3,3-tetramethyluronium hexafluorophosphate, 5.0 eq.], HOBt (1-hydroxybenzotriazole, 5.0 eq.) And DIEA (diisopropylethylamine, 10 eq.) For 2 minutes Treatment was activated.

This solution was added to the free amine on the resin and the coupling reaction proceeded for 1 hour simultaneously with vortex stirring. After washing with DMF solution, 9-fluorenylmethoxycarbonyl group was removed by 20% piperidine (twice: 1 × 10 min, 2 × 3 min) in DMF solution. The resin was washed with DMF (3 × 3 min) solution and the same reaction was repeated for the next amino acid. The linear peptide was cleaved from the resin by reacting with 5% triisopropylsilane (TIS) and 5% H ₂ O present in trifluoroacetic acid (TFA 2 mL per 100 mg of resin). The cocktail cut to precipitate the peptide was mixed with cold ether and then centrifuged.

5mm 입자 사이즈) 상에서의 RP- HPLC 분석에 의해 평가하였다.Preliminary reverse-phase HPLC (RP-HPLC) analysis was performed on a Vydac C ₁₈ column using a water / acetonitrile gradient of 0% to 90% in the presence of 0.05% TPA. The final purity (> 95%) of the peptide was determined by analytical Bidda C ₁₈ columns (4.6 mm × 250 mm, 300

Evaluation by RP-HPLC analysis on 5 mm particle size).

For sequence alignment (FIG. 1A) and phylogenetic analysis (see FIG. 1B), mastoparan MP-L, MP-X (V), MP-B, and MP-V1 are classified as typical mastoparans and are common. Share the motif.

On the other hand, bumblebee (Vespula mascarfaran (MP-V1) peptide having a sequence of SEQ ID NO: 1 isolated from the venom of vulgaris ) is an atypical mastoparan containing asparagine, the polar amino acid in an additional 15th sequence, consisting of 14 residues It is clearly distinguished from the typical mastoparan (see FIG. 1).

It was also found that MP-V1 has a seventh lysine, which is a polar side chain in the middle of the peptide, whereas typical mastoparan contains hydrophobic amino acids (see FIG. 1).

In addition, circular dichroism spectra were analyzed to investigate the secondary structure of polypeptides synthesized using JASCO J-715 spectropolar limiter (JASCO International, Tokyo, Japan).

The CD spectra of all the synthesized mastoparans showed a random coil (random-coil) form in aqueous solution (FIG. 1C). However, in the presence of 8 mM SDS and 40% TFE, the CD spectra of all mastoparans showed α-helical properties (FIG. 1C).

Percent α-helix, measured against mastoparan in different environments, is an aqueous solution, while mastoparan tends to form α-helix structures in the presence of 8 mM SDS or 40% TFE. Has been found to tend to form a random coil form (Table 1).

TABLE 1 Percent α-helix Characterization of Mastoparan V1 in Different Environments

In particular, of all mastoparans, MP-X (V) showed the highest α-helix percentage at 8mM SDS or 40% TFE, while MP-V1 had a slightly higher α-helix percentage compared to MP-L and MP-B. It was shown.

1-3. Preparation of Vaccine Using Mastoparan V1 as Adjuvant

Salmonella Gallinarum HJL 465 single colony prepared in Example 1-1 was independently injected into 200 mL of LB broth, incubated slowly at 37 ° C., and cultured at 600 nm until the optical density reached 0.3. .

The pathogen culture medium was treated with formalin to induce the death of pathogenic bacteria. Thereafter, the LB agar medium was placed in a 4000 g centrifuge and centrifuged for 30 minutes to kill dead cells, and resuspended in a PBS solution at a concentration of 3.0 x 10 ⁹ cells / mL. 20 μg / ml of mastoparan VI of SEQ ID NO: 1 prepared in Example 1-2 was added to the formalin-treated Bacillus vaccine prepared in Example 1-2, and used as a vaccine against pathogenic bacteria.

1-4. Positive and negative control

Formalin-treated vaccinated vaccine (3.0 × 10 ⁹ cells / mL concentration) commercially available from M Company was treated and used as a positive control group.

In addition, it did not contain the pathogenic dead or live bacteria, the same amount of PBS solution was used as a negative control group.

1-5. Statistical analysis

Pair-wise comparisons were performed by PHT test (post hoc turkey) using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). All values are expressed as mean ± SE for at least 3 independent experimental values. Statistical significance was determined at P <0.01 level.

Experimental Example 1. Immunogenicity Verification through Animal Model

Chickens raised in farms were divided into three groups, with 30 chickens in each group. Chickens in each group were divided into half and inoculated with the prime antigen at 6 weeks of age (0 WPPI) and orally at 3 weeks after 9 weeks of boost antigen (3 WPPI).

Chickens of group F were used as a control by intramuscular injection of sterile PBS solution, H-J group was diluted to 20 μL PBS solution at a concentration of 3.0 × 10 ⁹ CFU each of the vaccine prepared by the method of the above Example 15 each After intramuscular injection, Salmonella Gallinarum toxic strain of Example 1-1, which was isolated outdoors 3 weeks after the second inoculation, was cultured as a challenge strain, and the inoculum was diluted to a high concentration of 5 × 10 ⁸ CFU, followed by oral All chickens were inoculated (Table 2).

TABLE 2

Serum and fecal samples were taken to assess immune responses after 0, 3, and 6 week post prime inoculation (WPPI), respectively.

All animal experiments were performed under the approval of the Code of Ethics (CBU 2012-0017) of the Animal Ethics Committee of Chonbuk National University in accordance with the guidelines of the Korean Animal Protection Committee.

As a result, in F group treated with PBS solution, F chickens were observed to die from 8 days after challenge, and only 14% of 15 chickens survived after 14 days.

In addition, in the group I, a positive control group treated with commercially available formalin-treated vaccinated vaccines, chickens died from the 8th day after the challenge as in the negative control group F, and 15 chickens after 14 days. Only about 10% of the survivors were observed.

Unlike the negative and positive controls, in the J group treated with the vaccine prepared using the mastopararane peptide V1 of the present invention as an adjuvant, death occurred at 8 days after the challenge, but 14 days later. At the time point, 70% of the 15 chickens survived, and the results showed that the mastoparan peptide V1 acted as an adjuvant to directly enhance the vaccine effect against pathogenic bacteria.

Experimental Example 2 ELISA Immune Response

Salmonella Gallinarum Salmonella Gallinarum Outer membrane proteins (OMPs, outermembrane proteins) specific IgG (Invitrogen, Carlsbad, CA) to the vaccine prepared using Mastoparan V1 in the adjuvant of Examples 1-3 Titrated to serum samples collected from orally administered chicken, respectively, and analyzed using the ELISA method of Hu and Lee (Hur and Lee, 2011, Vaccine Immunolo. 18, 203-209), shown in FIG.

As a result, in the group J treated with the vaccine of the present invention prepared using mastopararan V1, plasma IgG concentrations specific for Salmonella outer membrane proteins (OMP) were increased by 3 WPPI. After 6 WPPI, it increased by 3 times compared to the negative control group treated with PBS solution, and increased 1.25 times compared to the positive control group treated with commercially available vaccinated vaccine.

Through the above results, the vaccine prepared using the mastopararane peptide V1 of the present invention as an adjuvant promotes the secretion of antibodies involved in the immune response through the humoral immune system, thereby enhancing the vaccine effect against pathogenic bacteria. Sikkim could be confirmed.

Experimental Example 3. Measurement of cytokines in splenocytes

After the experiment of Experimental Example 2 was finished, 5 chickens in each group were sacrificed to collect the spleen at 6 WPPI, and the spleen was aseptically collected according to the following method.

First, five chickens of each group were spleens collected aseptically at 2 weeks after the last vaccination and collected in RPMI 1640. Then, red blood cells were dissolved using 0.8% ammonium chloride (w / v) solution, and centrifuged at 380 × g and 4 ° C. for 10 minutes, and the precipitate was washed three times with sterile PBS. After the last centrifugation, the cells were resuspended with complete medium (RPMI 1640 supplemented with 100 IU / ml penicillin, 100 ug / ml streptomycin and 10% FCS). Calculate the number of cells to be cultured, react with each antigen for 72 hours, centrifuge at 380 × g, 4 ° C for 10 minutes, store the supernatant at -80 ° C, and use cytokine using ELISA assay. The concentration was measured.

As a result, as shown in Figure 4, in the J group treated with the vaccine of the present invention prepared using mastopararan V1 with adjuvants, the concentration of INF-γ is about four times higher than the negative control group treated with PBS solution The increase was 1.5 times higher than that of the positive control group treated with formalin-treated vaccinated vaccine.

Through the above results, it can be seen that the vaccine prepared using the mastoparan peptide V1 of the present invention as an adjuvant exhibits an excellent vaccine effect against pathogenic bacteria by secreting cytokines through a cellular immune system.

Experimental Example  4. Confirmation of defense through autopsy

Two weeks a day after challenge, mortality was checked twice a day, and all surviving chickens were examined at 2 weeks after challenge. It was confirmed whether the defense ability change.

As a result, as shown in Figure 5, it was confirmed that the size and weight of the liver is observed to be normal in the experimental group administered to the vaccine prepared by using the mastoparan peptide V1 of the present invention as an adjuvant (Fig. In contrast, in the positive control group treated with commercially available formalin-treated Bacillus vaccine (figure 5 hypertrophy), the size and weight of the liver were observed to increase.

Through the above results, it was confirmed that the adjuvant composition comprising the mastoparan peptide V1 of the present invention as an active ingredient can reduce the degree of infection or inflammation and exhibit high immune enhancing activity against pathogens.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> National Institute of Biological Resources          Gyeongnam National University Of Science And Technology Industry-Academic Cooperation Foundation          The Industrial-Academic Cooperation Group, Jeonbuk, Korea University          Komipharm International Co., Ltd <120> Adjuvant Compositions Comprising Mastoparan-V1 Peptide as Active          Ingredient <130> PN180020AN <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Vespula vulgaris <400> 1 Ile Asn Trp Lys Lys Ile Lys Ser Ile Ile Lys Ala Ala Met Asn   1 5 10 15

Claims (8)

Mastoparan MP-V1 peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
An adjuvant composition for vaccine against one or more Salmonella spp. Pathogenic bacteria selected from Salmonella Gallinarum, Salmonella Typhimurium, and Salmonella Enteritidis. delete delete delete The method of claim 1,
The mastoparan MP-V1 peptide is,
(Iii) a polynucleotide encoding the mastoparan MP-V1 protein of SEQ ID NO: 1; (Ii) an expression control sequence operably linked with the polynucleotide of (iii) to induce expression of the polynucleotide; And (iii) a recombinant expression vector comprising a transcription termination sequence is produced by the transformed cell into which the recombinant expression vector is introduced. (a) Vaccines against one or more Salmonella spp. pathogenic bacteria selected from Salmonella Gallinarum, Salmonella Typhimurium, and Salmonella Enteritidis, Salmonella Gallinarum, Vaccines against one or more Salmonella spp. Pathogenic bacteria selected from Salmonella Typhimurium, and Salmonella Enteritidis; And
(b) A vaccine composition comprising the adjuvant composition of any one of claims 1 to 5 as an active ingredient. delete delete

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