KR101615186B1 - Composition for enhancing protective efficaty of vaccine against tuberculosis and the method thereof - Google Patents

Abstract

The present invention relates to a composition for enhancing an immune effect and a method for pretreating a tuberculosis immunity for increasing the efficiency of a tuberculosis vaccine. According to the present invention, It can be applied to vaccines, which can have a great impact on the development of the pharmaceutical industry.

Description

Technical Field [0001] The present invention relates to a composition for enhancing an immune effect of a tuberculosis vaccine, and a method for enhancing an immune effect of the tuberculosis vaccine,

The present invention relates to a composition for enhancing the immunity effect and a method for pretreatment of tuberculosis immunity for increasing the efficiency of tuberculosis vaccine.

Tuberculosis is caused by Mycobacerium tuberculosis . Tuberculosis has a high incidence and mortality rate among the three WHO-designated infectious diseases, especially the domestic economic and national status. There are about 60 million active tuberculosis patients worldwide and it is estimated that 50 million to 100 million people are infected with tuberculosis every year. At least nine million new cases of tuberculosis occur each year and more than 1.5 million people die from tuberculosis. The incidence rate of tuberculosis is 146 deaths per 100,000 population and the death rate of tuberculosis is 49, which is the most common cause of death among infectious diseases. In recent years, the incidence of drug-resistant tuberculosis patients and HIV infection has become more serious. Approximately 50% of HIV infected people are infected by TB at the same time, and Mycobacterium tuberculosis promotes HIV proliferation and shortens life expectancy by a factor of 2 compared to other opportunistic infections. In addition, the rate of tuberculosis-related mortality was more than three times higher and the treatment effect was twice as low in HIV-positive tuberculosis patients. It is estimated that about 40% of Mycobacterium tuberculosis infection rate in Korea is infected with about 20 million people. Approximately 10% of these, 2 million people, are expected to become tuberculosis patients once in their lifetime. About 60% of people who die from infectious diseases in Korea are able to infer the seriousness of tuberculosis, and thus they are becoming serious problems in the 21st century's national health and welfare. In particular, the incidence and mortality rate of tuberculosis in Korea is the highest among the OECD countries since 1995.

Currently, tuberculosis is prevented using live bacteria of Bacillus Calmette-Guerin (BCG), a non-toxic strain of Mycobacterium bovis . However, there has been constant controversy over the efficacy of BCG, and there are significant differences in efficacy depending on country, region, and race. In addition, some countries, including the United States, prohibit the use of BCG as an efficacy issue. To date, there has been no development of a safer and more effective new vaccine to replace BCG. Therefore, one of the ways to prevent tuberculosis is to maximize the efficacy of the BCG vaccine currently in use.

The granulocyte receptor (Gr-1) is present in the form of a combination of Ly-6G and Ly-6C, and mainly plays a role in the differentiation of myeloid cells. Ly-6C is involved in the differentiation of monocytes, macrophages, and endothelial cells and is regulated by interferon gamma. In particular, Ly-6C is expressed in various cells such as bone-marrow cells, monocytes, macrophages, neutrophils, vascular endothelial cells, and specific T cells.

Accordingly, the present inventors have developed a method of enhancing the tuberculosis preventive effect of BCG vaccine by removing Gr-1 expressing cells including neutrophils expressing high level of Gr-1 when the BCG vaccine is inoculated The present invention has been completed.

It is an object of the present invention to provide a method for enhancing the efficacy of BCG vaccine for preventing tuberculosis by immunological intervention to remove Gr-1 expressing cells including neutrophils expressing Gr-1 in BCG vaccine .

In order to achieve the above object, the present invention provides a composition for enhancing an immune effect of a tuberculosis vaccine, which comprises a remover for a cell expressing a granulocyte receptor (Gr-1).

Also, the present invention provides a composition for pretreatment of tuberculosis immunization, comprising a remover for a cell expressing a granulocyte receptor (Gr-1).

In addition, the present invention provides a method for pretreatment of tuberculosis immunization, which comprises the step of administering to a subject a remover for graulocyte receptor (Gr-1) expressing cells.

In addition, the present invention provides a method for treating a cell, comprising the steps of: administering to a subject a remover for a cell expressing a granulocyte receptor (Gr-1); And a vaccine composition comprising a Mycobacterium bovis BCG (BCG) vaccine strain to the subject.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for enhancing an immune effect of a tuberculosis vaccine comprising a remover for a cell expressing a graulocyte receptor (Gr-1).

The present invention also relates to a composition for pretreatment of tuberculosis immunization comprising a remover for graulocyte receptor (Gr-1) expressing cells.

The composition for enhancing the immune effect of the tuberculosis vaccine may be an adjuvant used for enhancing the vaccine effect of the vaccine composition for the prevention of tuberculosis. It was confirmed that when the composition for enhancing immune effect of the present invention is used, the immune effect by BCG strain inoculation for tuberculosis prevention can be improved.

The anti-tuberculosis vaccine may preferably be a BCG vaccine by inoculation with a BCG strain.

The granulocyte receptor (Gr-1) exists in the form of a combination of Ly-6G and Ly-6C, and plays a role in the differentiation of myeloid cells.

The remover for the expression cells of Gr-1 includes all those capable of removing the cells expressing Gr-1, and may be an inhibitor for inhibiting the expression of Gr-1 in cells. Preferably an inhibitor that inhibits the expression of Ly-6G in the granulocyte receptor or a remover that removes cells expressing Ly-6G. Ly-6G is a kind of GPI-anchored protein. Since Ly-6G is expressed not only in neutrophils but also in eosinophils, differentiation monocytes or plasmacytoid dendritic cells, Inhibition of Ly-6G with monoclonal antibodies can selectively inhibit neutrophils more than Ly-6C inhibitor antibodies.

The inhibitor or remover may preferably be any one selected from the group consisting of anti-Gr-1 (clone RB6-8C5) antibody, anti-Ly-6G (clone 1A8) antibody and combinations thereof.

The above-mentioned Gr-1-expressing cell remover can most preferably use an anti-Gr-1 (clone RB6-8C5) antibody and more specifically can remove neutrophil expressing Gr-1 when the above antibody is used , The effect of the tuberculosis preventive composition can be further enhanced.

The Gr-1 expressing cell refers to a cell expressing a granulocyte receptor and is preferably a neutrophil (Neutrophils eosinophils, differentiating monocyte, plasmacytoid dendritic cells, and combinations thereof) And the like.

In the case of subcutaneous vaccination with BCG, the neutrophil is the most rapidly responding, and it plays a role of moving to the draining lymph nodes, and neutrophils express high level of Gr-1 on the cell surface, , It is confirmed that at least the large predominant cells and T cell activation are inhibited by inhibiting the killing of Mycobacterium tuberculosis. Thus, BCG vaccine for the prevention of tuberculosis through the method of temporarily removing neutrophils (Neutrophils) The immunization effect of the vaccine of the present invention can be improved. Specifically, by removing cells expressing Gr-1, including neutrophils, which are the fastest responding immune cells, it is possible to increase the survival rate of BCG, to inactivate large progenitor cells by Neutrophils, Can be prevented, and the prevention effect by the vaccination can be improved.

The composition for pretreatment for tuberculosis immunization refers to a composition for pretreatment which is administered prior to the administration of the composition for the prevention of tuberculosis to an individual. When the composition for pretreatment is used, the immunological effect of BCG vaccine for tuberculosis prevention is improved .

The composition for enhancing the immune effect of the tuberculosis vaccine or the composition for pretreatment for tuberculosis immunity may further comprise, as an active ingredient, a substance that solely includes a remover for the Gr-1-expressing cell as the active ingredient, or that is effective for the immunotherapy enhancement or immunization pretreatment And may further comprise additional components, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or subcomponents, depending on the formulation, the method of use and the intended use.

More particularly, the present invention relates to a pharmaceutical composition containing, in addition to the above-mentioned active ingredients, a nutrient, a vitamin, an electrolyte, a flavoring agent, a colorant, a thickening agent, a pectic acid and a salt thereof, alginic acid and a salt thereof, Glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. The carrier, excipient or diluent may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acicia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline In the group consisting of celluloses, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, But the present invention is not limited thereto. Any conventional carrier, excipient or diluent may be used. These components may be added to the active ingredient, that is, to the Gr-1 expressing cell remover, independently or in combination.

0.001 wt% to 99.9 wt%, preferably 0.1 wt% to 99 wt%, more preferably 1 wt% to 50 wt% of the effective ingredient, specifically, the Gr-1 expressing cell remover, based on the total weight of the composition. . The content of the additional component may be added in the range of 0.1% by weight to 20% by weight based on the total weight of the composition for enhancing immunity or the composition for pretreatment for tuberculosis immunization, but is not limited thereto.

In addition, when the composition is weakly formulated, it may further contain usual fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers or preservatives, can do.

The dose of the composition according to the present invention can be suitably selected by those skilled in the art in consideration of the administration method, the age, sex, and weight of the recipient, severity of disease, and the like. For example, the composition for immune enhancement or the composition for pretreatment of tuberculosis of the present invention may be administered at a dose of 0.0001 mg / kg to 1000 mg / kg, more preferably 0.01 mg / kg to 100 mg / kg, Even more effectively from 30 mg / kg to 50 mg / kg.

The present invention also relates to a method of pretreatment of tuberculin immunization comprising the step of administering to a subject a remover for graulocyte receptor (Gr-1) expressing cells.

The present invention also relates to a method for the treatment and / or prophylaxis of cancer, comprising the steps of: administering to a subject a scavenger for a graulocyte receptor (Gr-1) expressing cell; And a vaccine composition comprising a Mycobacterium bovis BCG (Vaccine BCG) vaccine strain to the subject.

When the removers of the granulocyte receptor (Gr-1) expressing cells are administered to an individual, there is an effect of enhancing the immune effect of the BCG vaccine for preventing tuberculosis.

It is preferable that the step of administering to a subject a remover for a cell expressing a granulocyte receptor (Gr-1) is performed before administration of the composition for the prevention of tuberculosis, more preferably, Day and 1 day after the administration of the composition for preventing tuberculosis, respectively. As described above, when the remover for the Gr-1 expressing cell is administered, there is an effect of improving the immunological effect of the composition for preventing tuberculosis.

The amount of the remodeling agent for the granulocyte receptor (Gr-1) expressing cells may be adjusted depending on the body weight of the individual to be administered and the administration method. For example, when administered to a mouse, 200-250 μg / mouse per mouse ≪ / RTI >

The subject includes all mammals including humans, preferably mammals other than humans.

 The removers of the granulocyte receptor (Gr-1) expressing cells can be administered through a respiratory or intraperitoneal injection, preferably by intravenous injection. In the case of intravenously injecting the above-mentioned remover, it is more effective to effectively remove Gr-1 expressing cells even when administering about half of the intraperitoneal injection dose.

The present invention relates to a composition for enhancing an immune effect of a tuberculosis vaccine, and the present invention can increase the immune effect upon use of the tuberculosis vaccine, and can be applied to various vaccines including BCG, It can have a big impact.

Fig. 1 is a schematic diagram showing a procedure for vaccination and infection experiment.
FIG. 2 is a graph showing the results of comparing the efficacy of a vaccine by counting the number of living Mycobacterium tuberculosis in the lung tissue after 8 weeks of attack after inoculation through the respiratory tract of the Mycobacterium tuberculosis Erdman strain. The abscissa of the graph represents the number of mycobacterium tuberculosis (CFU / lung) in the lung, and the abscissa of the graph represents the infection including vaccination alone, BCG vaccinated group (BCG), BCG and GR-1 (BCG + isotype Ab) treated with the composition containing the BCG and the isotype antibody and then attacked in the test group (BCG + GR-1).
FIG. 3 shows the results of comparing the efficacy of the vaccine by counting the number of living Mycobacterium tuberculosis in the spleen tissue at 8 weeks after the inoculation of the strain of Mycobacterium tuberculosis Erdman ( Mycobacterium tuberculosis Erdman) through the respiratory system. The vertical axis of the graph represents the number of tubercle bacilli in the spleen (CFU / Lung) and the abscissa of the graph represents an experiment in which the vaccination is only vaccination, BCG vaccinated group (BCG), BCG and GR-1 (BCG + isotype Ab) treated with the composition containing the BCG and the isotype antibody and then attacked in the test group (BCG + GR-1).
Fig. 4 shows histopathological results of pulmonary tissue at 8 weeks after respiratory infection with Mycobacterium tuberculosis Erdman ( Mycobacterium tuberculosis Erdman) strain. The rod bar means 1.0 mm and the magnification was observed at 20 times.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[Example 1] Preparation of mice, culture of Mycobacterium tuberculosis and BCG, immunization and evaluation of efficacy of the vaccine

1-1. mouse

A female C57BL / 6 mouse without a specific pathogen at 5-6 weeks of age was purchased from Japan SLC (Shijuoka, Japan) and raised in a confined space of BL-3 Bio Hazard Annunum Room at Yonsei University Medical Research Center. Mice were randomly given sterile, commercial mouse food and water.

1-2. Mycobacterium tuberculosis and BCG culture

Mycobacterium tuberculosis Erdman ( M. tuberculosis Erdman) and Mycobacterium bovis BCG str. Pastuer 1173P2 ( M. bovis BCG str., Pastuer 1173P2) were each cultured in 7H9-OADC broth for 15 days. The cultured strains were collected and then disrupted by gentle vortexing with 6 mm glass beads. The supernatant was collected after the agglomerates were submerged, and each was stored at -70 ° C. After thawing, live bacteria were counted by stepwise dilution plate culture in Middlebrook 7H11 agar (Difco, Detroit, MI, USA). In order to inoculate the Erdman strain in mice, the bacterial suspension was sonicated in a sonic bath, diluted with phosphate buffered saline (PBS) at pH 7.2, and used as the desired number of bacteria.

1-3. Assay of rat anti-monoclonal Ly-6G antibody and control isotype antibody

Neutrophils (neutrophils) generally express higher levels of Gr-1 in blood and tissue than other cells. In order to deplete cells expressing Gr-1 including neutrophils, rat anti-monoclonal Ly-6G (Cl-RB6-8C5) antibody and a control antibody (Rat ) IgG2b isotype antibody was purchased from BioXcell (Vwst Lebanon, NH, USA).

Prior to the experiment, mice were injected intraperitoneally with an anti-monoclonal Ly-6G antibody 200-250 ㎍ / mouse once or twice a day for a total of two times, and neutrophils in the tissues and blood were examined. As a result, (Neutrophils) are depletion.

1-4. Statistical analysis

Statistical significance was assessed using standard one-way ANOVA followed by Tukey's multiple comparison test or Dunnett's multiple comparison test. The statistical program used was GraphPad Prism version 5.00 (GraphPad Software, San Diego, Calif.). A p-value <0.05 was considered statistically significant.

[Example 2] Immunization

In the present invention, a total of four groups were experimented. Each group contained 6-8 mice. The group 1 was infected (challenge inoculation only), the group 2 was immunized only with BCG, the group 3 was immunized intraperitoneally with 250 μg of anti-monoclonal Ly-6G antibody one day before and after BCG immunization , Group 4 was an intraperitoneal injection of 250 ㎍ of control isotype antibody each day before and after BCG immunization.

The antibody was dissolved in sterile PBS according to the manufacturer's instructions, and stored at -80 ° C. Immunization test groups (groups 2, 3 and 4) were subcutaneously administered with BCG Pasteur 1173P2 (2 x 10 ⁵ CFU / mouse) strain (Fig. 1).

[Example 3] Identification of an immunological effect

3.1. Air infections of Mycobacterium tuberculosis and counting of bacterial numbers in the organ

The reduction in bacterial counts in mice immunized with mixed vaccines with preventive effects against M. tuberculosis infection was investigated. As described above, mice were immunized and infected at the 6th week after inoculation of 200-250 mycobacteria per mouse into a Glas-Col inhalation device (Terre Haute, IN) infection apparatus with highly pathogenic M. tuberculosis strain. At 8 weeks post infection, lung tissue was removed from each group of mice and the number of bacteria was determined. The number of bacteria was also confirmed for the spleen tissue in the same manner.

Specifically, the number of living bacteria in the tissues of each lung and spleen was confirmed by plating the whole tracheal homogenate suspension on the Middlebrook 7H11 agar in 5-fold serial dilutions. Colonies were counted after 3-4 weeks incubation at 37 &lt; 0 &gt; C and the results are shown in Fig. 2 and Fig. 3, calculated as the mean log10 CFU standard deviation per total lung tissue.

As shown in FIG. 2, the third group (BCG + Gr-1), which was administered with anti-monoclonal Ly-6G antibody intraperitoneally 200-250 占 퐂 / BCG), the number of mycobacteria living in lung tissue was significantly decreased (p <0.001).

3, a third group (BCG + Gr-1) in which anti-monoclonal Ly-6G antibody was administered intraperitoneally in a dose of 200-250 占 퐂 / mouse twice in a similar manner to lung tissue in spleen tissue (P <0.005) compared to the second group (BCG) immunized with BCG.

Therefore, it was confirmed that the BCG vaccine can significantly enhance the TB defense efficacy by removing immune cells such as Neutrophils expressing Gr-1 prior to BCG vaccination.

3-2. Histopathological analysis

Using the Glas-Col inhalation device (Terre Haute, IN) infection apparatus, the tuberculosis Erdman strain was infected with 200-250 mycobacteria per mouse into the respiratory tract, which is the general infection route of the Mycobacterium tuberculosis. At the 8th week, Were stored in 10% neutral-buffered formalin and fixed in a paraffin block. Fixed tissue was sectioned to 4-5 mm thickness and stained with hematoxylin and eosin (H & E). The result of the staining is shown in Fig.

As shown in FIG. 4, the third group in which neutrophils were removed by administering anti-monoclonal Ly-6G antibody intraperitoneally with a total of 200-250 占 퐂 / mouse together with BCG immunity was divided into a first group (an infected control group) And BCG-immunized group 2, the infiltration of the immune cells and the inflammation appeared to be less effective in the defense of tuberculosis.

Claims (6)

A composition for enhancing an immune effect of a tuberculosis vaccine, comprising an anti-Ly-6G antibody. delete The method according to claim 1,
The anti-Ly-6G antibody may be used to remove any one or more granulocyte receptor expressing cells selected from the group consisting of Neutrophils, eosinophils, differentiating mononuclear cells, plasmacytoid dendritic cells and combinations thereof Or a pharmaceutically acceptable salt thereof. A composition for pretreatment of tuberculin immunization comprising an anti-Ly-6G antibody. Tuberculosis vaccine adjuvant (ajuvant) comprising an anti-Ly-6G antibody. Administering an anti-Ly-6G antibody to an individual other than a human; And
And administering the tuberculosis vaccine to an individual other than the human.

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