KR102065848B1 - Composition for anti-influenza virus comprising Enterococcus canintestini - Google Patents

Abstract

The present invention is a pharmaceutical composition, vaccine composition and feed for the prevention and treatment of influenza virus infections containing a mixed agent of Enterococcus canintestini strain and Enterococcus canintestini and liquid sulfur extracted from the intestine of chicken It relates to a composition for addition.

Description

Composition for anti-influenza virus comprising Enterococcus canintestini

The present invention relates to an anti-influenza virus composition comprising a Enterococcus canintestini (KCTC No. 21021, Type Strain) strain and a mixture of sulfur and Enterococcus cannitestini strains.

More specifically, it shows an excellent antiviral effect by application or oral administration to the skin, and particularly contains a mixture of Enterococcus cannistinis strains and sulfur and Enterococcus carnistinis strains that induce inhibition and vaccine effects against influenza infection. The present invention relates to a pharmaceutical composition for preventing or treating influenza virus infection, a vaccine composition for prevention, and a composition for feed addition.

Influenza viruses are RNA viruses belonging to the orthomyxovirus and myxovirus genus (influenza virus genus), 80-120 μm long, many spherical, but also linear. It is largely divided into three types of A, B, and C. A type is divided into subtypes such as A0, A1, and A2, and B type is divided into subtypes such as B1, B1α, B1β, and B2. Hepatitis A virus is characterized by a slight change in antigen structure and sometimes a rapid change, which is called a discontinuous change. For example, infection with an influenza virus of a subtype of type A acquires immunity against the virus of this subtype, but a new type of virus is caused by a discontinuous change, thereby causing a pandemic. In particular, avian influenza (AI) caused by avian influenza virus infection is severely affected in poultry such as chickens, turkeys and ducks, and avian influenza virus has a large number of serum subtypes and is easily mutated. It is known that it is one of the most infectious livestock epidemics in terms of national defense because there is a wide variety of viruses in the wild birds of the ecosystem and they can pass without any obvious symptoms.

Currently, attenuated vaccines of influenza viruses are used as a prevention technique against such influenza. However, rapid vaccination against new influenza such as H1N1 influenza or avian influenza, which is a recent threat, has a long virus propagation time and difficult mass production, making it difficult to manufacture and at the same time, it is ineffective against variant viruses. . In addition, the conventional influenza vaccine is administered by injection mostly, the procedure is complicated, there are many disadvantages of time and place constraints, there is a disadvantage that the safety is a problem of high cost and direct virus administration.

In order to solve the disadvantages of the conventional influenza vaccine, there is a continuous demand for the development of new functional biologics having an antiviral effect against influenza.

The enterococcus group, on the other hand, lives in the intestine, mouth, vagina and urethra as normal intestinal flora. Although these enterococci have a low ability to cause disease, they are highly adaptable to the environment and can survive for a long time without being parasitic to living organisms. Enterococcus canintestini strains are Gram-positive bacteria, which are enterococci, which do not usually cause disease in humans, but are known to be involved in antibiotic resistance and in digestion and physiological activity. Enterococcus strains include Enterococcus pyecalis, Enterococcus pesium, E. faecium, Enterococcus avium, Enterococcus gallinarum, Enterococcus 19 species, including E. casseliflavus, are known, but no association with viral infections has been reported.

Sulfur is a widely used material for livestock feed, and it is known that it is possible to produce delicious livestock products by enriching cystine, cysteine and methionine, which are sulfur-containing amino acids that determine the taste of livestock products produced in livestock raising. To date, no impact on influenza virus infection has been reported.

Against this background, the present inventors have made diligent efforts to solve the shortcomings of the conventional influenza virus vaccines. As a result, the Enterococcus canintestini strain or a mixture of Enterococcus cannistinini strains and sulfur have an antiviral effect against the influenza virus. Confirmed to have completed the present invention.

An object of the present invention is to provide an Enterococcus canintestini strain that can induce antiviral effects against influenza infections.

Another object of the present invention is to provide a pharmaceutical composition and a vaccine composition containing a mixture of Enterococcus canintestini strain and sulfur which can prevent or treat influenza virus infection.

In addition, another object of the present invention is to provide a composition for feed addition containing a mixture of Enterococcus canintestini strain and sulfur.

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

According to one embodiment of the present invention, an Enterococcus canintestini (Accession No. KCTC No. 21021) strain resistant to influenza virus is provided.

According to one side, the influenza virus may be a type A influenza virus.

According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating influenza virus infection is provided, which contains a mixture of Enterococcus canintestini strain and liquid sulfur. .

According to one side, the concentration of the aqueous solution of the strain may be 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml.

According to another embodiment of the present invention, there is provided a vaccine composition for preventing influenza virus infection, which contains a mixture of Enterococcus canintestini strain and liquid sulfur.

According to one side, the concentration of the aqueous solution of the strain may be 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml.

According to one side, it may further comprise an adjuvant.

According to one side, the adjuvant is a complete Freund's adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), squalene (Squalene), squalane (Squalane), aluminum hydroxide (Aluminium hydroxide) ), At least one selected from the group consisting of aluminum phosphate and saponin.

According to one side, it may further comprise a pharmaceutically acceptable carrier.

According to another embodiment of the present invention, a composition for feed addition containing a mixture of Enterococcus canintestini strain and liquid sulfur is provided according to an embodiment of the present invention.

According to one side, the concentration of the aqueous solution of the strain may be 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml.

As described above, according to the present invention, the antiviral effect of the Enterococcus canintestini strain itself and the synergistic effect of the sulfur in the liquid form can achieve a better antiviral effect. In addition, the enterococcus cannistinis strain of the present invention exhibits a vaccine effect that can prevent reinfection by forming an immunity against influenza infections such as flu.

Furthermore, the enterococcus cannistinini strain of the present invention is cheaper and safer in terms of cost than the vaccine directly administered by the virus because it is a bacterium. In addition, oral administration of the strain, so microorganisms are formed in the individual, even in small doses may have the effect of preventing influenza virus infection or treating infectious diseases.

1 is a view showing the results of testing the effect of inhibiting the influenza virus growth in eggs by the mixed preparation of Enterococcus canintestini strain and sulfur.
Figure 2 is a view showing the results of the safety test by measuring the survival rate and weight loss in normal animals by a mixed preparation of Enterococcus canintestini strain and sulfur.
FIG. 3 is a diagram showing the results of blood expression of IL-12 cytokines by a mixed preparation of Enterococcus canintestini strain and sulfur.
4 is a diagram showing the results of testing the weight change (4a) and survival rate (4b) during oral administration of a mixed formulation of Enterococcus canintestini strain and sulfur infected with influenza virus.
Figure 5 is a test of the vaccine effect when the influenza virus reinfected with influenza virus after administration of the mixed agent of Enterococcus canintestini strain and sulfur of the present invention to the mice infected with influenza virus One result. 5 (a), 5 (b) and 5 (c) show the antibody production rate after infection (5a), weight change after infection (5b) and survival rate after infection (5c) of reinfected mice.
FIG. 6 is a diagram showing the results of testing the survival rate by oral administration of a mixed formulation of Enterococcus canintestini strain and sulfur infected with influenza virus. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of examples. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate that there is a feature, number, step, action, component, part, or combination thereof described on the specification, and one or more other features. It is to be understood that the present invention does not exclude the possibility of the presence or the addition of numbers, steps, operations, components, components, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art and shall not be construed in ideal or excessively formal meanings unless expressly defined in this application. Do not.

In addition, in the description with reference to the accompanying drawings, the same components regardless of reference numerals will be given the same reference numerals and redundant description thereof will be omitted. In the following description of the embodiment, when it is determined that the detailed description of the related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.

As used herein, the term "Enterococcus canintestini" refers to Gram-positive bacteria, which are intestinal inhabitants. Accession No. KCTC No. Refers to a strain of 21021.

As used herein, the term "prevention" means any action that inhibits or delays influenza virus infection by administration of a composition.

As used herein, the term "treatment" means any action that improves or beneficially alters the symptoms of an influenza virus infection by administration of the composition.

As used herein, the term "feed additive composition" refers to a substance added to the feed in order to improve the productivity or health of livestock.

As used herein, the term "feed" refers to a substance that supplies the organic or inorganic nutrients necessary to sustain livestock and produce milk, meat, eggs, fur, and the like.

According to one embodiment of the present invention, an Enterococcus canintestini (Accession No. KCTC No. 21021) strain resistant to influenza virus is provided.

Specifically, the influenza virus may be influenza A virus.

In the present invention, a mixture composition of sulfur and Enterococcus canintestini strains was prepared so that an optimal concentration of sulfur and Enterococcus canintestini may be suppressed. The aqueous strain solution may be mixed with sulfur at a concentration of 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml, more preferably at a concentration of 3 × 10 ⁹ cfu / ml.

Meanwhile, the Enterococcus canintestini strain is characterized by a strain of normal flora, not a resistant bacterium, and the Enterococcus canintestini strain is a strain having no pathogenicity.

According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating influenza virus infection is provided, which contains a mixture of Enterococcus canintestini strain and liquid sulfur. .

The pharmaceutical composition of the present invention may be used for any viral infection that may exhibit the antiviral effect, but is preferably used for the prevention or treatment of influenza virus infection, more preferably influenza A virus infection. .

The pharmaceutical composition may be administered with a pharmaceutical carrier generally used for antiviral treatment. Such carriers are used to formulate the pharmaceutical composition, and may generally include diluents, excipients, stabilizers, preservatives and the like.

The diluent may be a non-aqueous solvent such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or brine (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer) An aqueous solvent, such as these, etc. are mentioned, It is not limited to this.

The excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene, glycol, water , Ethanol and the like, but is not limited thereto.

The stabilizer may include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, and proteins such as animal, vegetable or microbial proteins such as milk powder, serum albumin and casein, but are not limited thereto. no.

The preservatives include, but are not limited to, thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, and the like.

The pharmaceutical composition of the present invention is administered in a therapeutically effective amount. The term "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, the effective amount being the severity, age, sex, time of administration, It can be determined according to the route of administration and the rate of excretion, the duration of treatment, factors including the concurrent drug and other factors well known in the medical field. For example, the adult Enterococcus canintestini strain of the present invention may be administered once from 1 mg to 1000 mg.

The pharmaceutical compositions may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

According to another embodiment of the present invention, there is provided a vaccine composition for preventing influenza virus infection, containing a mixture of Enterococcus canintestini strain and liquid sulfur.

Ingredients that may be further included in the vaccine composition are as described above in the pharmaceutical composition, and may further include an adjuvant to enhance the vaccine effect.

The adjuvant is a complete Freund's adjuvant (CFA), an incomplete Freund's adjuvant (IFA), squalene (Squalene), squalane (Squalane), aluminum hydroxide (aluminium hydroxide), aluminum phosphate ( Aluminum phosphate) and saponin may be one or more selected from the group consisting of, preferably, aluminum hydroxide and aluminum phosphate may be mixed in a gel form (Alum), but is not limited thereto.

The pharmaceutical composition or vaccine composition of the present invention may be administered to the human body via any general route as long as it can reach the desired tissue. Specifically, parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, but is not limited thereto. In addition, the composition may also be administered by any device in which the active agent may migrate to the target cell.

Parenteral administration means administration modes including intravenous, intramuscular, intraperitoneal, intrasternal, transdermal and intraarterial injection and infusion. Parenteral administration of the composition may be carried out under the desired purity in a pharmaceutically acceptable carrier, i.e., in a unit dosage form, which is a mixture of nontoxic to the receptor and compatible with other formulation ingredients at the concentrations and dosages employed. It may be desirable.

According to another embodiment of the present invention, a composition for feed addition containing a mixture of Enterococcus canintestini strain and liquid sulfur is provided according to an embodiment of the present invention.

Since the composition for feed addition of the present invention has antiviral efficacy against influenza virus, it can be expected to be effective in preventing or treating influenza virus infection when added to animal feed. The feed may be classified into various types according to nutritional value, main ingredient, distribution, moisture content and processing type, etc. In the present invention, the feed includes a feed, a rich feed, a supplement feed, a protein feed, a starch feed, a fat feed, and a fiber feed. B is not limited to this.

In the present invention, the composition for feed addition may be used for the production of livestock, poultry feed, specifically can be used for the production of poultry feed or pig feed. In addition, the composition for feed addition of the present invention may further include nucleotides, enzymes, trace components as well as other microorganisms to assist the growth of livestock necessary to fully supply the essential nutrients necessary for animal productivity or health promotion. .

For example, the nucleotides may be used alone or in combination with 5'-adenylic acid, xanthic acid, ribonucleic acid, deoxyribonucleic acid, and the like; Enzymes include arabinase, arabinofuranosidase, galactanase, pectinase, maltase, maltase, which are not provided from the three microorganisms. Invertase, lipase, and the like may be used alone or in combination; As a minor component, amino acid, calcium, phosphoric acid, organic acid, etc. can be used individually or in combination; Microorganisms include the Leuconostoc sp . Strain, the Weisella sp . Strain, the Enterococcus sp . Strain, the Streptomyces sp . Strain and the Nocardia genus. Nocardia sp .) Strains and the like can be used alone or in combination.

Animals that can feed the feed to which the composition for feed addition of the present invention is added are not particularly limited, but chickens, turkeys, ducks, geese, quails, pheasants, pigs, piglets, cattle, calves, sheep, goats and cats , Dog, etc.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.

Example 1 Preparation of Enterococcus canintestini Strain and Sulfur Mixture

The present formulation was prepared by mixing Enterococcus castinini strain aqueous solution and liquid sulfur (Ubiotech, Korea) in a ratio of 1: 1. The aqueous strain solution was prepared at a concentration of 3 * 10 ⁹ cfu / ml and liquid sulfur was mixed into the stock solution without any dilution to prepare a mixed formulation.

Example 2 Influenza Virus Inhibitory Effect Test in Cells of Enterococcus canintestini Strain and Sulfur Mixture

First, Enterococcus canintestini strain was cultured at 37 ° C. in Luria Bertani medium. A formulation was prepared in which sulfur was mixed with Enterococcus kanestistini strain cultured by the method described in Example 1. Cultured Enterococcus carnitestine strains, sulfur and the above mixed formulations were administered to the influenza A virus-infected eggs in an amount of 1 ml, respectively, to test the inhibitory effect of influenza A virus and are shown in FIG. 1. As a result, it was confirmed that the highest influenza type A virus inhibitory effect was observed in the formulation of sulfur and Enterococcus carnitestine strains.

Example 3 Infection Control Effect Test of Enterococcus canintestini Strain and Sulfur Mixture in Animal Model of Influenza Virus

After administration of the mixed formulation of Enterococcus canintestini strain and sulfur prepared in Example 1, the toxicity in the animal body by the strain was evaluated. Weight loss or mortality was evaluated, but no side effects were observed (FIG. 2).

In addition, it was confirmed by the ELISA method whether the antiviral cytokine interleukin 12 protein is secreted in the body. As a result, it was confirmed that the antiviral effect was expressed by secreting interleukin 12 protein by Enterococcus canintestini strain and sulfur mixed agent (FIG. 3).

Finally, in order to confirm the antiviral effect of the mixed formulations of the present invention on Salmonella strains, Balb / C mice were administered a mixed formulation of Enterococcus cannistinis strain and Enterococcus cannistinini strain and sulfur, and influenza A The virus was infected. The control group was nasally infected with 104 pfu of influenza A virus three days after bacterial treatment of normal model mice treated with only PBS solution without any Salmonella strain. In order to compare the effects of Enterococcus canintestini strain and sulfur mixed formulations with the control group, the weight change of mice due to infection was measured and shown in FIGS. 4A and 4B. Referring to Figure 4a and 4b, sulfur in the formulation, enterococos carnitesini in order of less weight loss, it can be seen that the survival rate is high.

Through the above results, it can be seen that the sulfur mixed agent of the present invention has an antiviral effect against influenza A virus while having less toxicity in the body.

Example 4 Vaccine Effect Test of Enterococcus canintestini Strain and Sulfur Mixture in Animal Model of Influenza Virus

After surviving 10 ⁴ pfu of influenza virus nasal to the surviving mouse group obtained in Example 3 and the control mice administered with PBS solution only, the survival rate and the antibody production was confirmed.

Figure 5a is a comparison of the degree of response of the IgG antibody to influenza A virus, it can be seen that the antibody reaction occurs by the mixed agent of the present invention.

Subsequently, 104 pfu of influenza virus was infected nasal to the surviving mouse group obtained in Example 3 and the control mice administered with only PBS solution, and then reinfected with influenza virus after 30 days for the surviving mice. And survival rate were compared.

As a result, as shown in Figures 5b and 5c, the group administered with the mixed agent of the present invention showed a slight weight change and at the same time showed a 100% survival rate.

These results suggest that the combination of Enterococcus canintestini strains and sulfur may not only prevent infection with influenza A virus but also have a vaccine effect.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus 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.

Claims (11)

delete delete A pharmaceutical composition for preventing or treating influenza A virus infection, which contains a mixture of Enterococcus canintestini strain (KCTC No.21021) and liquid sulfur.
The method of claim 3,
The concentration of the aqueous solution of the strain is 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml, pharmaceutical composition.
A vaccine composition for preventing influenza A virus infection, which contains a mixture of Enterococcus canintestini strain (KCTC No.21021) and liquid sulfur.
The method of claim 5,
The concentration of the aqueous solution of the strain is 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml, vaccine composition.
The method of claim 5,
A vaccine composition further comprising an adjuvant.
The method of claim 7, wherein
The adjuvant is a complete Freund's adjuvant (CFA), an incomplete Freund's adjuvant (IFA), squalene (Squalene), squalane (Squalane), aluminum hydroxide (aluminium hydroxide), aluminum phosphate ( One or more selected from the group consisting of aluminum phosphate) and saponin.
The method of claim 5,
A vaccine composition further comprising a pharmaceutically acceptable carrier.

A feed additive composition for preventing influenza A virus infection, which contains a mixture of Enterococcus canintestini strain (KCTC No.21021) and liquid sulfur.
The method of claim 10,
The concentration of the aqueous solution of the strain is 3 × 10 ⁸ cfu / ml to 3 × 10 ⁹ cfu / ml, type A influenza virus infection prevention feed additive composition.

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