KR102221244B1 - Vaccine composition for preventing tuberculosis comprising Mycobacterium anyangense strain - Google Patents

Abstract

The present invention relates to a vaccine composition for preventing tuberculosis comprising a Mycobacterium anyangense strain,
The vaccine composition for tuberculosis prevention based on Mycobacterium anyangense strain provided in one aspect of the present invention has superior stability and tuberculosis prevention effect compared to M. bovis BCG, which is currently used as a tuberculosis vaccine, so that the conventional tuberculosis vaccine It has the effect of securing a more excellent preventive effect by replacing it.

Description

Vaccine composition for preventing tuberculosis comprising Mycobacterium anyangense strain}

The present invention relates to a vaccine composition for preventing tuberculosis comprising a Mycobacterium anyangense strain.

Tuberculosis is a highly contagious infectious disease caused by Mycobacterium tuberculosis bacteria, and it is one of the three major infectious diseases determined by the WHO and has a high incidence and mortality rate. There are about 60 million active TB patients worldwide, and it is estimated that 50 million to 100 million people are infected with tuberculosis every year, at least 9 million new cases of tuberculosis occur annually, and more than 1.5 million people die from tuberculosis. The incidence rate of tuberculosis is 146 per 100,000 people, and the mortality rate of tuberculosis is 49 per 100,000 people, accounting for the most death among single infectious diseases, which remains a serious health problem worldwide. In recent years, due to the increasing number of refractory tuberculosis patients showing drug resistance and the increase in HIV infection, the outbreak pattern is becoming more serious. Currently, about 50% of HIV-infected people, or 15 million people, are simultaneously infected with Mycobacterium tuberculosis, and Mycobacterium tuberculosis promotes HIV growth, shortening the life expectancy by half compared to other opportunistic infections, which is more threatening to HIV-infected people. Has become. In addition, in the case of HIV-positive tuberculosis patients, it was reported that the mortality rate from tuberculosis was more than three times and the treatment effect was twice as low.

The rate of infection with Mycobacterium tuberculosis in Korea is about 40%, and it is estimated that about 20 million people have been infected with Mycobacterium tuberculosis. Of these, about 10%, or 2 million, are expected to become tuberculosis patients at least once in their lifetime. About 60% of people who die from infectious diseases in Korea can infer the severity of tuberculosis, which has emerged as a serious problem for national health and welfare in the 21st century. In particular, there are many tuberculosis bacteria of the Beijing family, which are highly pathogenic in Korea. Unlike other Mycobacterium tuberculosis, the Beijing strain is highly pathogenic and infectious, and the incidence of multidrug-resistant tuberculosis and reactivation tuberculosis is very high. Genetic fingerprinting of Mycobacterium tuberculosis in Korea revealed that the one with Beijing was predominantly present, and this genotype accounted for 18.4% of the total Mycobacterium tuberculosis.

On the other hand, Mycobacterium tuberculosis, which belongs to the Peking family and is highly infectious in Korea, is named K strain, and the K strain, a highly pathogenic tuberculosis bacteria in Korea, represents a specific pathogenesis mechanism capable of overcoming the initial innate immunity when infected with a mouse. It showed a growth rate corresponding to 10 times the infection amount of the H37Rv strain, the standard strain of Mycobacterium tuberculosis. The death of mice caused by infection with the Korean-type highly pathogenic M. tuberculosis K strain is more than twice as fast as that of mice infected with the H37Rv strain, the standard M. tuberculosis strain, and the number of bacteria at the time of death after infection is almost the same. As the immune pathology caused by it is considered to be very important as a pathogenesis mechanism, it was judged that the specific boosting of acquired immunity through preventive vaccines rather than innate immunity would be a key factor in defending the K strain, a highly pathogenic tuberculosis bacteria in Korea.

Currently, tuberculosis is mainly prevented using live bacteria of Bacillus Calmette-Guerin (BCG: Bacillus Calmette-Guerin), a non-toxic strain of Mycobacterium bovis (Republic of Korea Patent Publication No. 10-2019- 0014398). However, the safety and efficacy of BCG is controversial by region, country, and race, and the use of BCG is restricted in some countries, such as the United States. In addition, BCG is known to be unsuitable for use in HIV-infected or immunocompromised children. Accordingly, there is a need to develop a new, safer and more effective preventive vaccine to replace BCG.

Therefore, as a result of continuing research to develop a vaccine that is safe and can effectively prevent Mycobacterium tuberculosis, the inventors of the present invention have found that Mycobacterium anyangense , a fast-growing non-TB mycobacterium, KCTC29443) The present invention was completed by confirming that the strain itself or a composition using it exhibits excellent tuberculosis prevention effect.

Objects in one aspect of the present invention,

Compared to M. bovis BCG, which is currently used as a tuberculosis vaccine, it provides a vaccine composition for tuberculosis prevention and a pharmaceutical composition for tuberculosis prevention, which can replace M. bovis BCG because of its superior stability and tuberculosis prevention effect.

To achieve the above object,

One aspect of the present invention provides a vaccine composition for preventing tuberculosis comprising a Mycobacterium anyangense strain.

In addition, another aspect of the present invention provides a pharmaceutical composition for preventing tuberculosis comprising a Mycobacterium anyangense strain.

The vaccine composition for tuberculosis prevention based on Mycobacterium anyangense strain provided in one aspect of the present invention has superior stability and tuberculosis prevention effect compared to M. bovis BCG, which is currently used as a tuberculosis vaccine, so that the conventional tuberculosis vaccine It has the effect of securing a more excellent preventive effect by replacing it.

1 is a PBS for C57BL/6 mice, or ^{strains prepared in Example 1 (1×10 6} CFU), Example 2 (1×10 ⁷ CFU), and Comparative Example 1 (1×10 ⁶ CFU), respectively It is a graph showing the Mycobacterium tuberculosis CFU measured in the lungs (left graph) and spleen (right graph) of mice at 6 weeks after inoculation twice at intervals of 2 weeks and induction of Mycobacterium tuberculosis infection.
2 is a graph showing the results of comparative evaluation of the expression level of Th1 cytokine related to tuberculosis protective immunity.
3 is a graph showing the results of comparative evaluation of the amount of IgG2a related to Th1 cytokine expression.
4 is a graph showing the results of comparative evaluation of the ratio of IgG2a/IgG1 representing a protective immune response.
5 is a graph showing a comparison result of growth curves for each medium.
6 is a graph showing the results of comparing the survival amount related to safety in the tissues of nude mice.

Hereinafter, the present invention will be described in detail.

Tuberculosis is a disease caused by invasion and proliferation of the Mycobacterium tuberculosis complex (MTBC). Currently, M. bovis BCG is inoculated with live bacteria to induce interferon gamma, etc., thereby obtaining a tuberculosis prevention effect. M. bovis BCG is the only vaccine against tuberculosis, but it is difficult to deal with the strain due to its slow growth and community-forming properties. In addition, the effects vary according to region, race, and age, and are known to have no effect on adults. A new tuberculosis vaccine to replace this is being developed, but development is being attempted through genetic recombination via M. bovis BCG and/or non-tuberculosis mycobacterium, so stability problems still exist.

In order to solve this problem, an aspect of the present invention provides a vaccine composition for preventing tuberculosis comprising a Mycobacterium anyangense strain. The rapidly growing non-tuberculosis mycobacterium Mycobacterium anyangense strain may have been deposited as KCTC29443.

The Mycobacterium anyangense KCTC29443 is a strain that can replace the M.bovis BCG vaccine strain, which is used to prevent the incidence of tuberculosis and proliferation in the body during infection by the Mycobacterium tuberculosis complex (MTBC). to be. Mycobacterium anyangense (Mycobacterium anyangense) KCTC29443 itself or an immune-stimulating substance using it is injected into the body to induce an immune response, and through this, the effect of preventing tuberculosis can be obtained.

The Mycobacterium anyangense KCTC29443 grows faster than M.bovis BCG and does not use a recombinant gene, so there is no possibility of functional degradation due to genetic manipulation, excellent stability, and does not form a cluster during growth. It has the advantage of being easy to produce and handle.

Meanwhile, the tuberculosis may be tuberculosis caused by infection of the Korean-type highly pathogenic Mycobacterium tuberculosis K strain (Mycobacterium tuberculosis K strain).

More specifically, the tuberculosis is ocular tuberculosis, cutaneous tuberculosis, renal tuberculosis, epididymis tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug resistant tuberculosis, pulmonary tuberculosis, cholangitis, throat tuberculosis, pulmonary hemorrhage, breast tuberculosis and spinal tuberculosis. It may be one or more selected from the group consisting of.

The vaccine composition of the present invention can be administered alone, but preferably, it can be administered together with an adjuvant. Adjuvant is a substance that non-specifically promotes an immune response to an antigen in the initial activation process of immune cells.It is not an immunogen to a host, but refers to an agent or molecule that enhances immunity by increasing the activity of cells in the immune system (Warren et al., 1986, Annu. Rev. Immunol,. 4:369). Adjuvants that can be administered together with the vaccine composition of the present invention to enhance the immune response include any of a variety of adjuvants, and typical adjuvants include Freund adjuvant, aluminum compound, and Mura. Wheat dipeptide (muramyl dipeptide), phospholipid (LPS), monophosphoryl lipid A, or kuyl A, and the like are known. The adjuvant may be administered simultaneously with the vaccine composition or may be sequentially administered at intervals of time.

In addition, the vaccine composition of the present invention may further include a solvent, an excipient, and the like. The solvent includes physiological saline, distilled water, and the like, and the excipient includes, but is not limited to, aluminum phosphate, aluminum hydroxide, or aluminum potassium sulfate, and is not limited thereto. It may further include.

The vaccine composition of the present invention can be prepared by a method commonly used in the technical field to which the present invention belongs. The vaccine composition of the present invention can be prepared in an oral or parenteral formulation, and is preferably prepared as an injection solution as a parenteral formulation, and is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal or epidural ( eidural) route.

The vaccine composition of the present invention is administered in an immunologically effective amount. The immunologically effective amount refers to an amount sufficient to exhibit a tuberculosis prophylactic effect and an amount that does not cause side effects or serious or excessive immune reactions.The exact dose concentration varies depending on the specific immunogen to be administered, and Age, weight, health, sex, patient sensitivity to drugs, administration route, administration method, etc. can be easily determined by a person skilled in the art according to well-known factors in the medical field, and can be administered once to several times.

The vaccine composition for tuberculosis prevention based on Mycobacterium anyangense strain provided in one aspect of the present invention has superior stability and tuberculosis prevention effect compared to M. bovis BCG, which is currently used as a tuberculosis vaccine, so that the conventional tuberculosis vaccine There is an effect that can secure a more excellent preventive effect by replacing it, which is supported by the Examples and Experimental Examples to be described later.

In addition, the present invention provides a pharmaceutical composition for preventing tuberculosis comprising a Mycobacterium anyangense strain.

The pharmaceutical composition of the present invention may include one or more known active ingredients having a preventive effect of tuberculosis together with a Mycobacterium anyangense strain.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, and diluent commonly used in the preparation of a pharmaceutical composition. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

The pharmaceutical composition according to the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The composition of the present invention may be used alone for the prevention and treatment of tuberculosis, or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods of using a biological response modifier.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the examples and experimental examples to be described later are merely illustrative of the present invention in one aspect, and the present invention is not limited thereto.

<Example 1> Preparation of Live Mycobacterium Anyanzens KCTC29443

Mycobacterium Anyangens ( M. anyangense ) was prepared by culturing KCTC29443.

<Example 2> Heat-killed Mycobacterium Anyanzens KCTC29443 preparation

Heat-killed Mycobacterium Anyanzens KCTC29443 By applying high-temperature heat to kill live bacteria, live Mycobacterium Anyanzens KCTC29443 strain is sterilized through an autoclave method (121℃, 15 minutes), and then the final 1 * 10 ⁸ CFU using PBS. It was prepared by diluting to a concentration of /ml.

<Comparative Example 1> Mycobacterium bovis ( M. bovis ) BCG preparation

For comparison, M. bovis BCG, currently used as a tuberculosis vaccine, was prepared.

<Experimental Example 1> Mycobacterium tuberculosis prevention and immune effect evaluation

In order to evaluate the Mycobacterium Anyangens (M. anyangense ) KCTC29443 strain according to the present invention to protect against Mycobacterium tuberculosis infection, the following experiment was performed.

PBS for C57BL/6 mice, or ^{strains prepared in Example 1 (1×10 6} CFU), Example 2 (1×10 ⁷ CFU), and Comparative Example 1 (1×10 ⁶ CFU), each 2 weeks apart It was inoculated twice. More specifically, PBS, Examples 1, 2, and Comparative Example 1 are each 100 ul at a time using a 1 ml syringe by a flak skin subcutaneous injection method on the side of the mouse, a total of 2 times at intervals of 2 weeks ( 200 ul) was injected. The injection site was sterilized with alcohol and then inoculated, and after injection, the inoculation was confirmed by swelling of the injection site.

Afterwards, Mycobacterium tuberculosis was infected through air infection. At this time, TBG-014, which is found in a number of domestically, was used as the tuberculosis bacteria. The TBG-014 strain is a clinical isolate having the most representative genotype in Korea through the MIRU-VNTR genotyping method among clinical strains possessed by the National Institute of Health, and a strain classified as Beijing type based on the spoligotyping analysis method was used. . After shaking culture through Middlebrook 7H9 liquid medium (BD), a medium exclusively for Mycobacterium tuberculosis, a bacterial solution having an absorbance (OD600) of 1.0 to 1.5 was prepared as a single cell strain using a 10 um pore size syringe filter. The bacterial solution was stored in an ultra-low temperature freezer at -70° C. for 1 week, and the number of viable cells was measured by the agar plate method, and then used as a mouse challenge inoculation line.

At 6 weeks of infection with Mycobacterium tuberculosis through air infection, the mice were anesthetized and sacrificed, and the Mycobacterium tuberculosis CFU in the lung and spleen tissues of the mice was measured, and the results are shown in FIG.

1 is a PBS for C57BL/6 mice, or ^{strains prepared in Example 1 (1×10 6} CFU), Example 2 (1×10 ⁷ CFU), and Comparative Example 1 (1×10 ⁶ CFU), respectively It is a graph showing the Mycobacterium tuberculosis CFU measured in the lungs (left graph) and spleen (right graph) of mice at 6 weeks after inoculation twice at intervals of 2 weeks and induction of Mycobacterium tuberculosis infection.

As shown in Figure 1,

Live Mycobacterium Anyanzens KCTC29443 (Example 1), heat-killed Mycobacterium Anyanzens KCTC29443 (Example 2) inoculation groups according to the present invention are M. bovis BCG (Comparative Example), which is currently used as a tuberculosis vaccine (Comparative Example) 1) Compared with the vaccinated group, the immunity effect against Mycobacterium tuberculosis was similar or superior.

<Experimental Example 2> Evaluation of the expression level of Th1 cytokine related to tuberculosis protective immunity

After inducing Mycobacterium tuberculosis infection through the same procedure as in <Experimental Example 1>, the expression levels of Th1 cytokines (TNF, IFN-γ, IL-6) related to tuberculosis protective immunity in mice were evaluated. The results are shown in FIG. 2.

2 is a graph showing the results of comparative evaluation of the expression level of Th1 cytokine related to tuberculosis protective immunity. Media only group treated with cell culture medium (DMEM) only as negative control, positive control ConA (concavalin A, 5 ug/ml), and Mycobacterium tuberculosis antigen PPD (purified protein derivatives, 5 ug/ml) were It was treated with an antigen to measure a specific response.

As shown in Figure 2,

Live Mycobacterium Anyanzens KCTC29443 (Example 1), heat-killed Mycobacterium Anyanzens KCTC29443 (Example 2) inoculation groups according to the present invention are M. bovis BCG (Comparative Example), which is currently used as a tuberculosis vaccine (Comparative Example) 1) Compared with the vaccinated group, the expression level of Th1 cytokine related to tuberculosis protective immunity was relatively increased.

<Experimental Example 3> Evaluation of antibody distribution in mouse serum

After inducing Mycobacterium tuberculosis infection through the same procedure as in <Experimental Example 1>, the distribution of antibodies in mice was evaluated. The results are shown in FIGS. 3 and 4.

3 is a graph showing the results of comparative evaluation of the amount of IgG2a related to Th1 cytokine expression.

4 is a graph showing the results of comparative evaluation of the ratio of IgG2a/IgG1 representing a protective immune response.

As shown in Figures 3 and 4,

Live Mycobacterium Anyanzens KCTC29443 (Example 1) and heat-killed Mycobacterium Anyanzens KCTC29443 (Example 2) inoculation groups according to the present invention are M.bovis BCG (Comparative Example), which is currently used as a tuberculosis vaccine (Comparative Example) 1) Compared with the inoculation group, the amount of IgG2a and the ratio of IgG2a/IgG1 were relatively increased on the first day after infection with Mycobacterium tuberculosis.

The IgG2a related to Th1 cytokine expression and the IgG2a/IgG1 ratio showing a protective immune response are initially high, which means that an excellent protective ability against Mycobacterium tuberculosis is formed at an earlier time point compared to the conventional vaccine.

<Experimental Example 4> Measurement of the growth rate of Mycobacterium Anyanzens

5 shows the results of measuring the growth rate according to the presence or absence of serum components in the culture medium. The absorbance of the culture medium was measured by incubating for 20 days in Middlebrook 7H9 broth (7H9 broth) containing serum components, LB broth without serum components, Sauton's broth, and M9 broth. Rapid-growing Mycobacterium smegmatis ( M. smegmatis ) and Mycobacterium Anyanzens KCTC29443 showed similar rapid growth rates regardless of the presence or absence of serum components, whereas M. bovis BCG, the control strain, was related to the presence or absence of serum components. Delayed growth curve was shown without.

<Experimental Example 5> Safety evaluation using a nude mouse of Mycobacterium Anyanzens

Figure 6 shows the liver of mice 1, 14, and 28 days after ^{infection (1×10 6} CFU/mouse) intravenously with M. bovis BCG and Mycobacterium Anyanzens KCTC29443, respectively, in nude mice with weakened immunity. , The safety was evaluated by measuring the Mycobacterium tuberculosis CFU in lung and spleen tissues. In all organs, Mycobacterium Anyanzens KCTC29443 showed relatively lower CFU results compared to M. bovis BCG, particularly low CFU in lung tissue, and it was confirmed that the safety in nude mice was higher than that in the BCG control group.

Claims (5)

A vaccine composition for preventing tuberculosis comprising a strain of Mycobacterium anyangense.
The method of claim 1,
The Mycobacterium anyangense (Mycobacterium anyangense) strain is deposited as KCTC29443, the vaccine composition for preventing tuberculosis.
The method of claim 1,
The tuberculosis is tuberculosis caused by infection of the Korean-type highly pathogenic tuberculosis bacteria K strain ( Mycobacterium tuberculosis K strain), a vaccine composition for preventing tuberculosis.
The method of claim 1,
The tuberculosis is in the group consisting of ocular tuberculosis, cutaneous tuberculosis, kidney tuberculosis, epididymis tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug resistant tuberculosis, pulmonary tuberculosis, cholangiosclerosis, throat tuberculosis, pulmonary deficiency, breast tuberculosis, and spinal tuberculosis. A vaccine composition for preventing tuberculosis, characterized in that at least one selected.
Mycobacterium anyangense (Mycobacterium anyangense) a pharmaceutical composition for preventing tuberculosis comprising a strain.

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