KR20030012199A - Dna vaccine comprising cytokine gene as active ingredient - Google Patents

Abstract

PURPOSE: Provided are a DNA vaccine comprising as an active ingredient cytokine gene, more specifically, interleukin 18(IL-18) gene which inhibits the infection of Mycobacterium sp., and an inhibition method of the infection of Mycobacterium sp. using the same. CONSTITUTION: A transformed E. coli DH5@/TcCMVIL-18(KCTC 10019BP) is obtained by transformation with an expression vector TcCMVIL-18 capable of expressing interleukin 18(IL-18) gene in a mammal cell. The DNA vaccine for the inhibition of Mycobacterium sp. comprises the expression vector TcCMVIL-18 as an active ingredient, and pharmaceutically acceptable carrier.

Description

DNA Vaccine containing cytokine gene as an active ingredient {DNA Vaccine Comprising Cytokine Gene As Active Ingredient}

The present invention relates to a DNA vaccine comprising a cytokine gene. More specifically, the present invention provides a bacterium of the genus Mycobacterium using a DNA vaccine and an electric DNA vaccine containing an interleukin 18 (IL-18) gene, which is known to inhibit infection of the genus Mycobacterium. The present invention relates to a method of inhibiting infection.

Mycobacterium avium complex (MAC) is known as a causative agent of bacterial infectious diseases spread in AIDS patients. As a major means for inhibiting the infection of MAC, cytokines such as interferon are used. Especially, cytokines known to be effective for MAC are known as γ-interferon (IFN-γ) and activated CD4 + T cells. It is known to be produced from neutral killer (NK) cells. Recently, interleukin-18 (IL-18), a known inducer of IFN-γ, has been found to be a new cytokine. IL-18 promotes the production of IFN-γ from activated CD4 + T cells and neutral killer cells. It is also known to provide an important defense against infectious pathogens.

In general, for the treatment of MAC infection, recombinant IFN-γ is clinically used, but the process of purifying recombinant IFN-γ requires a lot of costs, and the administration effect of IFN-γ is maintained for a short time. Therefore, the disadvantage that the frequent administration should be carried out, but the situation does not find a solution.

Therefore, there is a constant need to develop a method for effectively treating IFN- [gamma] in the treatment of MAC infection.

Therefore, as a result of earnest research efforts to develop a method for effectively treating IFN-γ in the treatment of MAC infection, when a DNA vaccine containing an expression vector containing a gene encoding an interleukin 18 is administered to an MAC infected animal. The present invention was completed by confirming that the infection of MAC was effectively suppressed by showing a similar effect to the treatment with IFN-γ.

After all, the main object of the present invention is to provide a DNA vaccine comprising a gene encoding interleukin 18.

Another object of the present invention is to provide a method for inhibiting infection of MAC using an electric DNA vaccine.

1 is a diagram showing a gene map of the expression vector TcCMVIL-18.

2 is a graph showing the effect of the expression vector on the production of IFN-γ.

3A is a graph showing the percentage ratio of measured cell lysis after 3 weeks of infection.

3B is a graph showing the percentage ratio of measured cell lysis after 8 weeks of infection.

DNA vaccine containing the gene encoding the interleukin 18 (IL-18) of the present invention is an expression vector capable of expressing the IL-18 gene in mammalian cells as an active ingredient, and includes a pharmaceutically acceptable carrier do.

The present inventors studied to develop a method for effectively treating IFN- [gamma], which is known as a cytokine that effectively inhibits MAC infection, in an animal infected with MAC. Rather than treating IFN- [gamma] directly with an animal infected with MAC, Although treatment with IL-18, which is known to induce the production of IFN-γ, has been shown to be effective in indirectly inhibiting MAC infection, even with recombinant IL-18, the high production costs and frequent administration of The problem could not be solved fundamentally. Therefore, an expression vector capable of expressing the IL-18 gene in an animal cell was prepared and administered to the animal infected with MAC, thereby attempting to express recombinant IL-18 in the infected animal cell.

In one embodiment of the present invention, the IL-18 gene of the mouse is obtained by PCR using genomic DNA obtained from the tail of the mouse as a template, which includes an SV origin, a CMV promoter, a β-lactamase gene, and the like. An expression vector TcCMVIL-18 capable of expressing IL-18 was cloned into an eukaryotic cell expression vector, and then introduced into E. coli to prepare a transformant, followed by culturing the transformant to obtain a large amount of the expression vector. It was. At this time, the expression vector containing the IL-18 gene is not particularly limited, and any expression vector capable of expressing the IL-18 gene in eukaryotic cells may be used.

When the expression vector was administered to the mice infected with MAC, it was found that killing the mice infected with MAC effectively inhibited the proliferation of MAC, and this effect was maintained for about 8 weeks or more. The effect of this expression vector was confirmed that IL-18 expressed by the expression vector is induced by inducing overproduction of IFN-γ, it was confirmed that the level of NO ₂ ^- additionally increased. Therefore, it was found that the infection of MAC can be effectively suppressed by using the DNA vaccine containing the gene encoding IL-18 of the present invention.

Accordingly, the present inventors named Escherichia coli transformed with the electric expression vector TcCMVIL-18 as Escherichia coli DH5 @ / TcCMVIL-18 (Esherichia coli DH5 @ / TcCMVIL-18). The gene bank was deposited with the accession number KCTC 10019BP.

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 of ordinary skill 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. .

Example 1 Preparation of IL-18 Expression Vector

Primer 1: 5'-TTTCCACGATGTGAAACTTTGGCCGACTT-3 '(SEQ ID NO: 1) and Primer 2: 5'-GAAGATCTCTAACTTTGATGTAAGTT-3' (SEQ ID NO: 2) containing a portion of the cDNA construct of mouse IL-18 from the mouse cDNA library (Clontech, USA) PCR was performed to obtain an IL-18 gene containing the desired restriction enzyme site, which was then expressed in mammalian expression vector "TcCMVOVA" containing SV40 origin (Lim YS et al ., Immunology 94: 135-141). , 1998) to construct the IL-18 expression vector "TcCMVIL-18" (see Figure 1). Electrolyzed TcCMVIL-18 was introduced into Escherichia coli to prepare transformed Escherichia coli, which was cultured in an LB plate medium containing 50 ug / ml ampicillin, and then, the cultured Escherichia coli was pulverized and cesium chloride density gradient (cesium) was obtained therefrom. chloride density gradient) to obtain a large amount of purified TcCMVIL-18 DNA by centrifugation. The endotoxin level of the purified DNA was measured using the Limulus amebocyte lysate assay kit (Biowhittaker, Walkersville, MD) to measure the cytotoxicity of the obtained DNA. It was found that the following.

The expression vector TcCMVIL-18 thus prepared was introduced into COS-7 cells (ATCC CRL-1651, American Type Cell Collection, USA), transformed, cultured for 3 days, and then centrifuged to obtain a culture supernatant. CNBr-Sepharose 4B resin bound with IL-18 antibody was treated and centrifuged to immunoprecipitate. Electroprecipitated resin was washed three times with 50 mM Tris buffer (pH 8.0) containing 0.1% (v / v) Tween 20, eluted with SDS-PAGE sampling buffer and electrophoresed, IL-18 Western blots were performed using the antibodies. As a result, it was found that IL-18 is normally expressed by the expression vector introduced into the COS-7 cells.

Accordingly, the present inventors named Escherichia coli transformed with the electric expression vector TcCMVIL-18 as Escherichia coli DH5 @ / TcCMVIL-18 (Esherichia coli DH5 @ / TcCMVIL-18). The gene bank was deposited with the accession number KCTC 10019 BP.

Example 2 Pharmacological effect of IL-18 expression vector

The expression vector prepared in Example 1 was injected into the mouse muscle, and the degree of infection of the pathogen of the injected mice, the amount of cytokines produced in the body, and the cytotoxicity of the electric expression vector were measured.

Example 2-1 : Inhibition Effect of Expression Vector

20, 100 or 300 μg of the expression vector TcCMVIL-18 DNA prepared in Example 1 was dissolved in 100 μl of 0.85% (w / v) physiological saline and intramuscularly injected into the quadriceps of each mouse using a 28-gauge insulin syringe. The experimental group was prepared, and the control group was prepared by intramuscular injection of the same concentration of TcCMV in the same manner, respectively, and the non-treated group was prepared. After 1 day, each experimental group, the control group and the untreated group were infected with MAC, and after 3 and 8 weeks of infection, the lungs of each experimental group, the control group and the untreated group were removed and homogenized by adding saline solution. Afterwards, the cells were plated on a solid medium (Middlebrook 7H11 agar plate, Difco Lab., USA) and incubated at 37 ° C. for 7 days to determine the number of colonies generated (see Table 1).

Infection inhibition effect of expression vector (log scale) Treatment group Number of colonies after 3 weeks Number of colonies after 8 weeks Survival Non-injection group 5.8 ± 0.2 7.6 ± 0.3 62 ± 4.7 Control (20 µg) 5.6 ± 0.3 7.0 ± 0.2 ND Control (100 µg) 5.0 ± 0.1 6.1 ± 0.3 73 ± 3.5 Control (300 µg) 5.1 ± 0.1 6.0 ± 0.2 77 ± 4.7 Experimental group (20 µg) 5.9 ± 0.2 7.9 ± 0.4 ND Experimental group (100 µg) 5.7 ± 0.1 7.3 ± 0.2 ND Experimental group (300 µg) 6.0 ± 0.2 7.6 ± 0.1 61 ± 2.2

As shown in Table 1, it was found that the infection rate of the experimental group was significantly lower than the control group after 3 and 8 weeks of infection.

Example 2-2 : Cytokine Production Enhancement Effect of Expression Vector

300 μg of the expression vector TcCMVIL-18 was dissolved in 100 μl of 0.85% (w / v) saline solution and intramuscularly injected into the quadriceps of the mice using a 28-gauge insulin syringe to prepare experimental groups. A control group was prepared by intramuscular injection and a non-treated group was prepared. After 1 day, each experimental group, control group and untreated group were infected with MAC, and immediately after infection, 1, 2, 4, 6 and 8 weeks later, lungs of each experimental group, control group and untreated group were removed, After homogenization by adding saline solution, lung cells were selected from the homogenate of each lung, and 5 × 10 ⁵ lung cells were collected at 1 μg / ml Concanavalin A (ConA, Sigma Chem. Co., USA). Incubated for 72 hours in RPMI 1640 medium (Gibco BRL, USA) containing the. The supernatant was obtained by centrifugation of the culture, and the level of IFN-γ contained in the supernatant was measured using a sandwich ELISA method: the antibody coated on the plate was subjected to rat anti-mouse IFN-γ (HB170). A bitinylate rat anti-mouse IFN- [gamma] (XMG 1.2) is used as the bitinylate secondary antibody, and the minimum limit for detection of IFN- [gamma] is 125 pg / ml (see FIG. 2). Figure 2 is a graph showing the effect of the expression vector on the production of IFN-γ (■) is an untreated group, (□) is an experimental group, (iii) represents a control group. As shown in Figure 2, the experimental group was found to maintain a high level of IFN-γ for 8 weeks compared to the control and untreated group.

Example 2-3 : NO ₂ ^- Production Enhancement Effect of Expression Vector

The experimental group was prepared by dissolving 100 or 300 μg of the expression vector TcCMVIL-18 prepared in Example 1 in 100 μl of 0.85% (w / v) physiological saline and then intramuscularly injection into the quadriceps of mice using a 28-gauge insulin syringe. In the same concentration, TcCMV was intramuscularly injected in the same manner to prepare a control group, and an untreated group was prepared. After 1 day, each experimental group, control group and untreated group were infected with MAC, and the non-infected group which was neither injected nor infected was prepared separately. After 3 and 8 weeks of infection, the lungs of each experimental group, control group, untreated group and uninfected group were removed, homogenized by adding saline solution, and lung cells were selected from the homogenate of each lung. ⁵ × 10 lung cells were incubated for 72 hours in RPMI 1640 medium containing 1 μg / ml concanavalin A. The supernatant was obtained by centrifuging the culture, and 50 μl of each supernatant, 100 μl of 1% (w / v) sulfonylamide and 0.1% (w / v) N-1-naphthyl-ethylenediamine dihydrochloride 100 μl of dissolved 2.5% (v / v) polyphosphoric acid was mixed at room temperature for 5 minutes, and the absorbance was measured at 540 nm using a spectrophotometer, and then compared with a standard curve of Na (NO ₂ ) as a standard. , NO ₂ ^- level of each sample was measured (see Table 2).

NO ₂ ^- level of each sample Treatment group 3 weeks past After 8 weeks Uninfected group <2.0 <2.0 Untreated group 35.2 ± 4.8 58.8 ± 1.3 Control (100 µg) 29.8 ± 6.0 60.4 ± 5.2 Control (300 µg) 41.3 ± 2.8 66.7 ± 4.9 Experimental group (100 µg) 113.7 ± 7.2 210.5 ± 13.7 Experimental group (300 µg) 128.3 ± 5.7 207.2 ± 18.8

As shown in Table 2, even after 8 weeks after the infection, the NO ₂ ^- level of the experimental group was found to maintain a higher level than the control, uninfected and untreated group. Increasing NO ₂ ^- levels are associated with the activation of macrophages, one of the important killing mechanisms of infected microorganisms. These results indicate that TcCMVIL-18 DNA injection continues to activate macrophages. James, SL, Microbiol. Rev., 59: 533-547, 1995; James SL & Nacy, C., Curr. Opin., Immunol., 5: 518-523, 1993).

Example 2-4 Measurement of Cytotoxicity of Expression Vectors on Infected Cells

100 μg of the expression vector TcCMVIL-18 DNA prepared in Example 1 was dissolved in 100 μl of 0.85% (w / v) physiological saline, and then injected into the quadriceps of the mouse to prepare an experimental group, and the same concentration of TcCMV was prepared in the same manner. A control group was prepared by intramuscular injection, and an untreated group was prepared. After 1 day, the experimental, control and untreated groups were infected with MAC, and after 3 and 8 weeks of infection, the lungs of the experimental, control and untreated groups were removed. Subsequently, the cells were homogenized by the addition of physiological saline, and the lung cells were selected from the homogenates of each lung, and then P815 cells (macrophage-sensitive cells) labeled with 5 × 10 ⁴ lung cells and ⁵¹ Cr radioisotopes at different ratios. ) Is mixed at 2: 1, 10: 1, 25: 1 or 50: 1 (cell number), reacted at 37 ° C. in a CO ₂ incubator for 4 hours, and the amount of ⁵¹ Cr released into the culture was measured. , As percentage of specific cytolysis (% cytolysis) (see FIGS. 3A and 3B). The percentage ratio of cell lysis is then calculated as follows:

Figure 3a is a graph showing the percentage ratio of cell lysis measured after 3 weeks of infection, Figure 3b is a graph showing the percentage ratio of cell lysis measured after 8 weeks of infection, (■) is the experimental group, (▲) The control group and (●) represent the untreated group and the mixing ratio represents the ratio of lung cells to P815 cells. As shown in FIG. 3A, the experimental group after 3 weeks showed much higher cytotoxicity than the control group, and as shown in FIG. 3B, it was confirmed that the high cytotoxicity of the experimental group was maintained even after 8 weeks.

Example 2-5 Analysis of Mechanism of Action of DNA Vaccine

It was confirmed that the production of increased IFN-γ by TcCMVIL-18 DNA injection was induced from anti-mycobacterial activity: that is, after each muscle injection of isotype antibodies similar to IFN-γ antibodies to mice , 300μg TcCMVIL-18 DNA dissolved in 100μl of 0.85% (w / v) saline was intramuscularly injected into the quadriceps of each mouse to prepare the experimental group, and the same concentration of TcCMV was intramuscularly injected to prepare the control group. The non-treated group was not prepared with injection of antibodies. After 1 day, each experimental group was infected with MAC. Subsequently, the antibody was injected intramuscularly once every 2 days for 6 days, after which the muscle was injected once every week. Three weeks after MAC infection, lungs were removed from the control, experimental and untreated groups to carry out the experiments of Examples 2-1 to 2-3 (see Table 3).

Effect of IFN-γ Antibodies on MAC Inhibition of Expression Vectors Treatment group Log scale NO ₂ ^- level (uM) % Cytotoxicity (1:50) Untreated group 5.9 ± 0.3 30.6 ± 4.8 13.8 ± 3.2 Control (IFN-γ Antibody) 6.1 ± 0.2 22.3 ± 5.7 18.1 ± 2.5 Control (isotype antibody) 5.8 ± 0.2 31.8 ± 6.4 12.8 ± 1.6 Untreated group 5.1 ± 0.1 120.8 ± 5.5 29.7 ± 3.3 Experimental group (IFN-γ antibody) 6.2 ± 0.3 36.2 ± 5.1 12.6 ± 1.8 Experimental group (isotype antibody) 5.0 ± 0.2 108.7 ± 6.7 33.6 ± 4.2

As shown in Table 3 above, when treated with IFN- [gamma] antibodies, the anti-mycobacterial activity degree observed in the experimental group was significantly reduced. The number of bacteria in the experimental mouse lungs treated with IFN- [gamma] antibodies was higher than that in experimental mice treated with isotype antibodies. In addition, the degree of NO ₂ ⁻ production and cytotoxicity to P815 cells by lung cells of mice treated with IFN-γ antibody was significantly lower than that of mice treated with isotype antibody. Therefore, it could be seen that the mycobacterium infection inhibition effect by the expression vector is due to increased NO ₂ ⁻ production, cytotoxicity, etc., which is due to the increased IFN-γ by the expression vector.

Administration method and formulation

A DNA vaccine containing an expression vector TcCMVIL-18 capable of expressing the IL-18 gene of the present invention in a mammalian cell as an active ingredient and a pharmaceutically acceptable carrier can be administered by parenteral injection. Pharmaceutically acceptable carriers are preferably isotonic aqueous solutions or suspensions and contain auxiliaries (eg, preservatives, stabilizers, wetting or emulsifier solution promoters, salts for controlling osmotic pressure and / or buffers). In addition, they may contain other therapeutically valuable substances.

Dosage

The dosage of the DNA vaccine of the present invention is different depending on the age, weight, and the degree of symptoms of the patient, but in the case of adults (based on 60 kg body weight), it is preferable to administer 200 to 400 mg once a day parenterally. It may be appropriately determined by the experience of those skilled in the art.

Acute Toxicity Test

In order to examine the acute toxicity of DNA vaccine in the present invention, the abdominal cavity of a DNA vaccine in male C57BL / 6 mice injected and hayeotneun determining the LD ₅₀ value by observing the number of death of mice over a 7 days after administration of the bar, LD ₅₀ value Was about 70 mg / kg. Therefore, it was found that the anticancer agent containing the expression vector TcCMVIL-18 as an active ingredient capable of expressing the IL-18 gene of the present invention in a mammalian cell in the range of the effective amount indicated above was found to be a sufficiently safe drug.

As described and demonstrated in detail above, the present invention is to inhibit the infection of bacteria of Mycobacterium by using a DNA vaccine and an electric DNA vaccine containing the interleukin 18 gene known to inhibit the infection of bacteria of Mycobacterium Provide a method. When the DNA vaccine of the present invention is administered to an animal infected with bacteria of Mycobacterium spp., Proliferation of bacteria of Mycobacterium spp. Infected with mice is suppressed for a long time, and the level of IFN-γ and the level of NO ₂ ⁻ are increased. Therefore, it can be widely used in the treatment of cancer and the treatment of bacterial infectious diseases.

Claims (9)

Expression vector TcCMVIL-18 capable of expressing IL-18 gene in mammalian cells. Escherichia coli DH5 @ / TcCMVIL-18 (KCTC 10019BP) transformed with the expression vector TcCMVIL-18 of claim 1. A DNA vaccine for inhibiting bacterial infection, comprising as an active ingredient an expression vector capable of expressing the IL-18 gene in a mammalian cell, and comprising a pharmaceutically acceptable carrier. The method of claim 3, wherein Expression vector is TcCMVIL-18 DNA vaccine for inhibiting bacterial infection. The method of claim 3, wherein Pharmaceutically acceptable carriers are aqueous isotonic solutions, suspensions or auxiliaries. Characterized by DNA vaccine for inhibiting bacterial infection. The method of claim 5, Adjuvants include preservatives, stabilizers, wetting agents or emulsifier solution accelerators, Salt and / or buffer for osmotic pressure control DNA vaccine for inhibiting bacterial infection. The method of claim 3, wherein The bacterium is characterized in that the microorganism of the genus Mycobacterium DNA vaccine for inhibiting bacterial infection. A method of inhibiting infection of a bacterium of Mycobacterium using a DNA vaccine, comprising administering the DNA vaccine of claim 3 to a mammal not a human infected with the bacterium of Mycobacterium. The method of claim 8, DNA vaccine comprising the expression vector TcCMVIL-18 A method of inhibiting the infection of bacteria of Mycobacterium using a DNA vaccine.