KR20040074190A - Therapeutic dna vaccine for tuberculosis and pharmaceutical composition containing the same - Google Patents

Abstract

PURPOSE: A therapeutic DNA vaccine for tuberculosis and a pharmaceutical composition containing the same are provided, thereby continuously inducing immune response, so that possibility of tuberculosis relapse can be reduced, and reducing the therapy period when it is used in combination with the conventional chemotherapy. CONSTITUTION: The therapeutic DNA vaccine for tuberculosis comprises the signal sequence of Herpes simplex virus type I glycoprotein D(gD) having the nucleotide sequence set forth in SEQ ID NO:1 and Ag85A gene having the nucleotide sequence set forth in SEQ ID NO:2, wherein the therapeutic DNA vaccine for tuberculosis is a vector pGX10-Ag85A(KCTC 10418BP) which is prepared by inserting the signal sequence of Herpes simplex virus type I glycoprotein D(gD) of SEQ ID NO:1 and Ag85A gene of SEQ ID NO:2 into a vector pGX10. The pharmaceutical composition contains the therapeutic DNA vaccine for tuberculosis and tuberculosis treating agent selected from isoniazid(INH), pyrazinamide(PZA), rifampin, streptomycin and ethanmbutol.

Description

DNA vaccine for treating tuberculosis and pharmaceutical composition containing same {Therapeutic DNA vaccine for tuberculosis and pharmaceutical composition containing the same}

The present invention relates to a tuberculosis vaccine and a composition for treating tuberculosis containing the same, and more particularly, to a tuberculosis DNA vaccine comprising tuberculosis antigen DNA and a composition for treating tuberculosis comprising the same.

One third of the world's population (about 1.9 billion people) is infected with Mycobacterium tuberculosis , and between 8 million and 10 million new cases occur each year, and an estimated 3 million people die from tuberculosis every year. . In developed countries, tuberculosis patients are also spreading due to the spread of HIV infection. The mortality rate from tuberculosis has risen again since the 1990s, mainly due to the increased incidence of multidrug-resistant Mycobacterium tuberculosis and the increased frequency of tuberculosis due to the spread of HIV. There are currently about 50 million patients with multi-drug resistant tuberculosis (MDR-TB) worldwide, but the treatment efficiency is only 50%. In addition, there are currently about 6 million HIV / TB simultaneous infections worldwide, and most of them are estimated to die from tuberculosis.

Mycobacterium tuberculosis is a rod-shaped bacillus with a very slow growth rate. It is an acid-resistant bacterium that can withstand dry conditions, strong acids or alkalis. The path of infection of tuberculosis is that bacteria mixed in the sputum drops of tuberculosis patients float in the air and inhale when they breathe into the lungs. Most of them heal naturally, but 5-10% of people develop tuberculosis and develop when their body's immunity is reduced due to complications such as overwork, undernutrition and diabetes (Kochi et al ., Lancet 1997, 350: 142; Toossi and Ellner, Pathogenesis of tuberculosis.In: Friedman LN (ed) .Tuberculosis: current concepts and treatment.CRC Press: New York, 2001, pp. 19-47). Therefore, it is very important to develop prophylactic and therapeutic vaccines to protect people who are not infected with TB or who are infected with TB.

Current treatment and prevention of tuberculosis include chemotherapy (Chemotheraphy) and BCG vaccination (Rieder HL, International Union Against Tuberculosis and Lung Disease: Paris, 2002). Although chemotherapy, especially DOTS (directly observed therapy, short course), is a very effective method for treating tuberculosis with a tuberculosis treatment rate of about 85%, it requires continuous administration for more than six months and has significant side effects. Not only is it costly, it is only used in 12% of patients with active disease because of its opacity for multidrug-resistant tuberculosis and its inability to rule out the possibility of relapses due to its opaque acquisition of persistent immunity (Broekmans JE, Control strategies and program management.In: Porter JDH, McAdam KPWJ (ed) .Tuberculosis: Back to the Future.John Wiley and Sons: New York, 1994, pp. 171-192; Chaulk et al ., J Am Med Assoc, 1998, 279: 943-948; Mitchison DA, Tubercle , 1985, 66: 219-225).

BCG is the most attenuated live vaccine that attenuates the virulence of a bacterium called Mycobacterium bovis and is the most widely used worldwide and appears to be effective in protecting children from widespread tuberculosis. It is known. It is also ineffective against the intrinsic reactivation of the latent M. tuberculosis . In addition, irregular chemotherapy may be a major cause of multi-drug resistant tuberculosis, and therefore, shortening the duration of chemotherapy with a new drug or therapeutic vaccine is a very important factor in the treatment of tuberculosis.

To date, DNA vaccines have been tried as new prophylactic tuberculosis vaccines and have been shown to be useful in animal models. Recently, prophylactic DNA vaccines expressing M. tuberculosis antigen or cytokines have been in the spotlight due to their efficiency and long-term sustained effects in animal models. For example, prophylactic DNA vaccines expressing antigens 85A, 85B, 65 kDa heat shock protein or PstS-3 have been found to be effective in limiting the growth of M. tuberculosis in mice (Denis Oet al. Infect Immun1998;66:1527-1533; Huygen ket al. Nat med1996;2:893-898; Lozes eet al. Vaccine1997;15:830-833; Kamath ATet al.Infect Immun1999;67:1702-1707; Tascon REet al.Nat med1996;2:888-892; Lowrie dbet al.Vaccine1997;15:834-838; Tanghe Aet al. J Immunol1999;162:1113-1119). In addition, DNA vaccination has proven to be very effective in establishing cellular immune responses, including cytotoxic T lymphocyte (CTL) and Th1 responses, which are known to be essential for obtaining effective anti-mycobacterial immunity in mice and humans. (Bonato VLet al.,Infect Immun,1998,66:169-175).

However, there are very few therapeutic vaccine candidates required for patients infected with tuberculosis compared with such effective prophylactic vaccine candidates. M. vaccae and cytokine IFN-γ or IL-12 have been studied in the Cornell model of M. tuberculosis latent infection, but the effectiveness of the candidate vaccine prevents the recurrence of tuberculosis. Was not enough (Corlan Eet al., Respir Med,1997,91:21-29; Holland SM, Dorman SE.,J Invest Med,1998,46(Suppl.) 218A; Holland SMet al., N Engl J Med,1994,330:1348-1355). DNA vaccines for therapeutic tuberculosis vaccines have been actively studied recently, and the effects have been reported to be insignificant (Turner J.et al,.Infect. Immun.,2000,68: 1706-1709; Repique CJet al.,Infect.Immun.,2002,70: 3318-3323). However, the use of therapeutic DNA vaccines expressing hsp65 after chemotherapy has been shown to be very effective in preventing reactivation of M. tuberculosis (Lowrie DB).et al., Vaccine,2000,18:1712-1716; Lowrie dbet al.,Nature,1999,400:269-271).

In tuberculosis patients, macrophages infected with M. tuberculosis are surrounded by active T cells (Andersen P,Trends immunol2001;22:160-168). Both CD4 + and CD8 + T cells are known to lyse infected macrophages that express mycobacterial antigens (Boom et al.,Infect Immun,1991,59:2737-2743; Kaufmann SH.,Annu Rev Immunol,1993,11:129-163; Tan jset al., J Immunol,1997,159:290-297). In particular, analysis of immunological mechanisms showed that antigen-specific CD8 + / CD4- / CD44 producing IFN-γ^highImmune responses and Th1 immune responses induced by memory type CTL have been shown to be required for the action of tuberculosis therapeutic vaccines (Bonato VLet al.,Infect Immun, 1998,66:169-175). Thus, immunotherapies that can increase the efficacy of the immune system in infected patients can be important adjuncts for effectively performing antibacterial chemotherapy. Several leading studies have shown that recombinant IL-12 protein in combination with antibacterial drugs eliminates Leishmania major infection or M. avium infection compared to treatment with cytokines or drugs alone. Induction (Naborset al.,Proc Natl Acad Sci USA,1995,92:3142-3146.1; Doherty TM, Sher A.,J Immunol,1998,160:5428-5435). However, these cytokine therapies are very toxic when applied clinically. Therefore, it is very important to develop a combination treatment method that is both effective and nontoxic for tuberculosis.

Accordingly, the present inventors have been trying to develop a DNA vaccine that is effective in preventing or treating tuberculosis and can increase immunity efficiency and reduce the duration of chemotherapy when applied simultaneously with chemotherapy. The treatment of the vaccine not only reduces the reactivation of latent M. tuberculosis, but also increases the production of INF-γ, revealing that the DNA vaccine can be usefully used to treat tuberculosis. Completed.

It is an object of the present invention to provide a tuberculosis DNA vaccine comprising the Ag85A gene and a tuberculosis treatment pharmaceutical composition containing the DNA vaccine and tuberculosis therapeutic agent as an active ingredient.

Figure 1 is an electrophoresis picture of immunoprecipitation analysis of the expression of Ag85A protein after transduction of pGX10-Ag85A vector into COS7 cells.

Figure 2 is a histopathological picture of the lung observed at various time periods after aerosol infection in a latent infected mouse model of tuberculosis.

A: 4 weeks after aerosol infection,

B: 18 weeks after aerosol infection,

C: 42 weeks after aerosol infection

3 is a schematic diagram showing the timing of chemotherapy (INH and PZA treatment), DNA vaccine treatment, and bacterial counting time after aerosol infection in a latent infection mouse model of tuberculosis ( A ) and 30 after aerosol infection in a latent infection mouse model of tuberculosis. At week 14, 14 weeks after the end of the combination of DNA vaccine and chemotherapy, mouse spleen cells produced CF (culture filtrate) -specific IFN-γ production ( B ) and Ag85A protein-specific IFN-γ production ( C ). The graph shown.

Figure 4 is a graph showing the immunotherapy effect when treated with a combination of chemotherapy and DNA vaccine in the latent infection model of tuberculosis through bacterial counts.

A: Lungs of mice 30 weeks after aerosol infection,

B: spleen of mice 30 weeks after aerosol infection,

C: Lungs of mice 42 weeks after aerosol infection,

D: Spleen of 42 weeks after aerosol infection

In order to achieve the above object, the present invention provides a tuberculosis DNA vaccine comprising a signal sequence of the herpes simplex virus type I glycoprotein D (gD) and the Ag85A gene.

The present invention also provides a pharmaceutical composition for treating tuberculosis containing the tuberculosis DNA vaccine and tuberculosis therapeutic agent as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a tuberculosis DNA vaccine comprising the signal sequence of the herpes simplex virus type I glycoprotein D (gD) and the Ag85A gene.

Preferably, the signal sequence of the herpes simplex virus type I glycoprotein D (gD) is set forth in SEQ ID NO: 1, and the Ag85A gene is preferably set forth in SEQ ID NO: 2.

The tuberculosis DNA vaccine of the present invention is a DNA vaccine comprising a signal sequence of the herpes simplex virus type I glycoprotein D (gD) and an Ag85A gene, wherein the genes are preferably inserted into a DNA vaccine vector. In a preferred embodiment of the present invention, the pGX10 vector was used as a DNA vaccine vector, and the pGX10 vector was a polyvinyl tail, SV40 enhancer after CMV promoter, tripartite leader sequence (TPL), simian virus 40 (SV40), and the like. ) Is a structure connected in series. An expression vector was prepared by inserting the signal sequence of the herpes simplex virus type I glycoprotein D (gD) described in SEQ ID NO: 1 and the Ag85A gene described in SEQ ID NO: 2 into the pGX10 vector and calling it "pGX10-Ag85A". It was deposited on February 3, 2003, by the Korea Institute of Biotechnology and Gene Bank (Accession Number; KCTC 10418BP).

The present invention also provides a pharmaceutical composition for treating tuberculosis containing the DNA vaccine and tuberculosis therapeutic agent as an active ingredient.

In the above, the tuberculosis treatment agent is preferably selected from the group consisting of isoniazid (INH), pyrazinamide (PZA), rifampin, streptomycin and ethambutol, and is INH or PZA. More preferred.

It has been reported that the number of antigen-specific T cells producing IFN- [gamma] during infection of M. tuberculosis is greatly reduced (Ormeet al.,J Immunol,1993,151:518-525; Zhang Met al., Infect Immun,1995,63:3231-3234). In a preferred embodiment of the present invention, the mouse spleen cells treated with IL-12 modified DNA vector, DNA vector itself or Ag85A DNA vector with chemotherapy using INH and PZA after 30 weeks of TB infection in a latent infection model of mice IFN-γ production was confirmed after stimulation with CF (culture filtrate) or Ag85A protein. As a result, when the IL-12 modified DNA vector was used in combination with the INH and PZA chemotherapy, IFN-γ production was compared with the control group treated with Ag85A DNA or the DNA vector itself together with the chemotherapy with INH and PZA. Was high (3ofBReference). In the case of stimulation by CF, there was no high level of IFN-γ induction in the Ag85A DNA-treated mice group of the present invention. Stimulation with Ag85A protein, on the other hand, showed higher levels of IFN-γ production in the mice immunized with Ag85A DNA compared to the DNA vector itself or mice immunized with IL-12 modified DNA.3ofCReference). The results indicate that an Ag85A-specific Th1 immune response can be generated by Ag85A DNA immunity, indicating the potential for inhibiting reactivation of M. tuberculosis. In addition, not only CF-specific IFN-γ production but also Ag85A-specific IFN-γ production were higher in mice immunized with IL-12 modified DNA than in mice immunized with DNA vector itself. Ag85-specific IFN- [gamma] induction is increased by immunization of, and the antigen-specific T-helper immune response generated after tuberculosis infection is converted to the type biased by Th-12 response by IL-12 modified DNA immunity. This means that there is a possibility of inhibiting reactivation of tubercosis.

In the present invention, the tuberculosis treatment effect of Ag85A DNA or IL-12 modified DNA vaccine, which was performed in combination treatment with INH and PZA for 3 months, was examined. Mice treated only with INH and PZA without DNA immunity at 30 weeks after infection with M. tuberculosis, Mycobacterium tuberculosis, were found in 80% and 100% of the lung and spleen, respectively. when used in combination therapy was 20% and the lung of mice, 40% of the mice are found to M. tuberculosis in the spleen (see B in Figure 4 a and Figure 4). As described above, simultaneous injection of the IL-12 modified DNA of the present invention is effective in inducing a Th1 immune response. Thus, it was found that the effective depletion of immune cells against M. tuberculosis antigen by IL-12N220L DNA and the conversion to Th1 by IL-12N220L DNA may reduce the reactivation of M. tuberculosis. Can be. In addition, when treated with Ag85A DNA vaccine organisms survive in the lung was not found, the spleen-to-20% of the mice only at the M. Bell System claw has been found (see B in Figure 4 A and Figure 4). These results indicate that the DNA vaccine expressing Ag85A or IL-12 modified protein in combination chemotherapy of INH and PZA inhibits the reactivation of M. tuberculosis inactive in the latent infection model of tuberculosis. It can be seen that it can be used as an adjuvant.

To determine whether the DNA vaccine of the present invention continuously affects the reactivation of M. tuberculosis, bacteria were counted at 42 weeks of M. tuberculosis infection, and 80% of the lungs and spleens of mice treated only with INH and PZA, respectively. and at 60% of the M. Bell-to-close-cis it has been found (see C and D, Table 1 of Fig. 4 in Fig. 4). Treatment with the DNA vector itself alone showed a 60% reactivation rate in the lungs and spleen. On the other hand, M. tuberculosis was found in 40% of the lungs and spleen of mice treated with the IL-12 modified DNA of the present invention. It has a sustained effect (Ha SJ et al ., Nat Biotechnol, 2002, 20: 381-386). Similar to the pattern seen at 30 weeks post infection, mice treated with Ag85A DNA vaccine showed reactivation rates of M. tuberculosis only in 20% of lung and spleen.

Therefore, the tuberculosis DNA vaccine in combination with the chemotherapy of the present invention can be used in the treatment of tuberculosis because it can continuously suppress the tuberculosis bacteria than chemotherapy itself, and at the same time, it is useful for the treatment of tuberculosis by shortening the treatment period Can be used.

An “immunologically effective” dose of the pharmaceutical composition of the invention is a dose suitable for eliciting an immune response. The particular dose depends on the age, weight and medical symptoms of the mammal to be treated and the method of administration. Proper doses will be readily determined by one skilled in the art. The vaccine composition may optionally be administered in a pharmaceutically or physiologically acceptable vehicle, such as physiological saline or phosphate buffered saline or ethanol polyols such as glycerol or propylene glycol. Small amounts of detergent may also be included to enhance vaccine stability. Compositions of the present invention may comprise vegetable oils or emulsions thereof, surfactants such as hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-diooctadecyl- N′-N′bis (2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and pluronic polyols; Polyamines such as pyran, dextransulfate, poly IC, carbopol; Peptides such as muramyl dipeptide, dimethylglycine, tuftsin; Immune stimulating complexes; Oil emulsions; Lipopolysaccharides such as MPLR (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Montana); And additional adjuvants such as mineral gels. The compositions of the invention are also incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolide, or ISCOMS (immunostimulatory complex) Supplementary active ingredients can also be used.

In addition, the pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be performed according to conventional methods such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection. In order to formulate into a parenteral formulation, the DNA vaccine and tuberculosis agent are mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated, but generally in adults one day The DNA vaccine can be administered once to several times in an amount of 0.1 to 0.2 mg per kg of body weight, preferably 0.02 to 0.1 mg.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Preparation of DNA Vaccine

<1-1> Preparation of Expression Vector for Ag85A DNA Vaccine Preparation

To prepare an effective tuberculosis DNA vaccine, the inventors made a fusion cassette in which the signal sequence of herpes simplex virus type I glycoprotein D (gD) described in SEQ ID NO: 1 was linked to the Ag85A gene described in SEQ ID NO: 2. .

The inventors prepared a vector in which the ampicillin resistance gene of the pTV2 vector (Lee et al., J Virol 1998; 72: 8430-8436) was substituted with the kanamycin resistance gene as a vector for inserting the fusion cassette prepared as described above. It was named "pGX10" afterwards. After preparing the expression vector inserting the fusion cassette prepared above into the pGX10 vector, it was named "pGX10-Ag85A" and deposited on February 3, 2003 at the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10418BP). ).

To determine whether the Ag85A gene is expressed as a protein, transfecting the pGX10-Ag85A DNA vector to COS7 cells in a general manner, and then using mouse anti-Ag85A serum prepared using Ag85A protein according to a conventional method. Radio-immunoprecipitation analysis was performed.

As a result, the Ag85A protein was not expressed in cells transfected with the pGX10 vector, which is an empty vector containing no Ag85A gene, whereas the band of Ag85A protein was expressed in cells transduced with the pGX10-Ag85A vector inserted with the Ag85A gene. Was confirmed (see FIG. 1 ).

<1-2> Preparation of Expression Vector for IL-12 Modified DNA Vaccine Preparation

Mutation of the codon encoding Asn-222 of the p40 subunit of IL-12 to Leu-222 results in lower secretion of IL-12p40 subunit, a natural antagonist of IL-12 (Ha SJet al.,Nat Biotechnol,2002,20:381-386), and also immunizing the IL-12 modified DNA with HCV (hepatitis C virus) E2 DNA promotes sustained E2-specific CTL responses and IFN-γ-producing CD8 + T cell responses compared to normal IL-12 DNA (Ha SJet al.,Nat Biotechnol,2002,20:381-386). Thus, the present inventors prepared a DNA construct that modified the DNA of IL-12 to prepare another tuberculosis DNA vaccine. Specifically, the preparation of the IL-12 DNA construct is as follows. Unidirectional serial linkage of IL-12p35 (mp35) in mice, internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV), and IL-12p40 (mp40) cDNA in mice were carried out in one direction. An expression vector was prepared by inserting it into the pGX10 vector of Example <1-1> and calling it "pGX10-mp35 / IRES / mp40 (pGX10-IL-12)". Next, in order to mutate the Asn-220 N-glycosylation position of IL-12p40 (mp40) from the vector containing the IL-12 DNA construct prepared above, Leu was isolated from the Asu-220 using a PCR method. Amino acid substitutions were carried out at -220 (Holland SMet al., N Engl J Med,1994,330:1348-1355). As a result, an IL-12 modified DNA expression vector was prepared in which pGX10-IL-12 was replaced by Leu-220 with Asn-220 in the mp40 region, which was expressed as "pGX10-mp35 / IRES / mp40-N220L (pGX10-IL- 12N220L) ".

Example 2 Establishment of Latent Infection Model

<2-1> Preparation of Antigen

The antigen for infection to be used in the latent infection model was prepared using Mycobacterium tuberculosis, Mycobacterium tuberculosis. Specifically, M tuberculosis H37Rv (ATCC 27294) was grown for 10 days with surface pellicles on Sauton medium, and the surface biomass was collected and slowly shaken with 3 mm glass beads and broken. The supernatant was collected after aggregation had settled, and the aliquot was stored at -70 ° C. After dissolving the stored aliquots, viable organisms were counted by serial dilution plating in Middlebrook 7H11 agar (Difco, Detroit, Mich.). To inoculate M. tuberculosis in mice, the suspension of the strain prepared above was briefly sonicated and diluted to 250,000 pfu / ml using PBS.

<2-2> Latent Infection

We made some modifications to the Cornell model (McCune et al., J Exp Med 1966; 123: 445-468) to latently infect Mycobacterium tuberculosis. Specifically, 5-6 week old female C57BL / 6J mice (SLC, Shijuoka, Japan) were placed in an inhalation chamber (Glas-Col, Terre Haute, Ind.) Of the airborne infection apparatus. 10 ml was exposed for 60 minutes with M. tuberculosis H37Rv 250,000 pfu / ml prepared in>. When the above process is performed, human tuberculosis bacteria are naturally transmitted to the animal through air.

M. tuberculosis was counted in the following manner one day after exposure of Mycobacterium tuberculosis to confirm that the mice were infected with Mycobacterium tuberculosis. Mice were euthanized with carbon dioxide and homogenized lungs and spleens in saline with 0.05% Tween 90 dissolved. Tissue suspensions were diluted 10-fold and 100 μl of each dilution was plated on M7H11 agar plates (Difco, Detroit, Mich.) And incubated at 37 ° C. for 3-4 weeks. After counting M. tuberculosis colonies on each plate, the results are expressed in log ₁₀ colony forming units (CFU).

As a result, it was confirmed that about 200 M. tuberculosis were delivered to the lung.

In addition, histopathological examination was performed to confirm that the delivery of M. tuberculosis identified above to the lung results in tissue change. Specifically, 4 weeks after the aerosol infection of M. tuberculosis, lung tissue was extracted, fixed in 10% formalin, and paraffin was inserted over several hours. 2-3 μm sections were made from paraffin-inserted tissue blocks and stained with haematoxylin and eosin.

As a result, most of the air zones of the lungs were filled with monocytes ( A in FIG. 2 ), and at this point counting tuberculosis bacteria that survived the lungs and spleens of infected mice, 6.69 ± 0.20 cfu (log) ₁₀ ) and 5.17 ± 0.11 cfu (log ₁₀ ) were detected.

<2-3> Chemotherapy in Latent Infections

After confirming that latent infection was successfully performed in Example <2-2>, chemotherapy was performed for the latent infection model of the present invention. 4 weeks after aerosol infection in mice in the same manner as in Example <2-2>, 25 mg / kg / day of isoniazid (ison HZ) (hereinafter referred to as "INH") in food (Sigma-Aldrich, Co. , MO, USA) and 1,000 mg / kg / day of pyrazinamide (hereinafter referred to as "PZA") (Sigma-Aldrich, Co., MO, USA) were added to mice for 3 months. After complete treatment with INH and PZA, the histopathological examination method described in Example <2-2> was performed to confirm that the infected tuberculosis was treated in the mice, and the tuberculosis bacteria were counted.

As a result, two weeks after complete treatment with INH and PZA for three months (18 weeks after aerosol infection), most infiltrated cells disappeared and only a clean air area remained ( B in FIG. 2 ), No viable M. tuberculosis was observed. However, after 42 weeks of infection, ie 26 weeks after discontinuation of INH and PZA treatment, there was a sign of lymphocyte infiltration ( C in FIG. 2 ). In addition, most of M. tuberculosis were reactivated in the spleen of 4 mice (80%) of the lungs or all 5 mice (100%) of the 5 mice 30 weeks after infection. Similarly, viable Mycobacterium tuberculosis was detected in most mice at 42 weeks post infection ( Table 1 ). From the above results, it can be seen that the infection model established by carrying this air reflects the existing Cornell model results (McCune et al., J Exp Med 1966; 123: 445-468). In contrast, when treated with INH and PZA for 6 months, no detectable number of bacteria was observed at 30 and 42 weeks after infection ( Table 1 ).

Treatment site and duration lungs spleen 30 Weeks 42 Weeks all 30 Weeks 42 Weeks all INH + PZA (6 months) 0/5 ^a 0/5 0/10 0/5 0/5 0/10 INH + PZA (3 months) 4/5 4/5 8/10 5/5 3/5 8/10 INH + PZA (3 months) + control DNA 3/5 3/5 6/10 3/5 3/5 6/10 INH + PZA (3 months) + IL-12N220L DNA 1/5 2/5 3/10 2/5 2/5 4/10 INH + PZA (3 months) + Ag85A DNA 0/5 1/5 1/10 1/5 1/5 2/10

a: Number of mice with regrowth of M. tuberculosis / Number of mice infected with H37Rv M. tuberculosis

Example 3 Antigen-Specific IFN-γ Production by IL-12N220L or Ag85A DNA Vaccine

In order to observe whether the DNA vaccine has a therapeutic effect during the chemotherapy in a latent infection model of mice, the inventors performed 3 months with INH and PZA 4 weeks after the aerosol infection as in the method of Example 2 above. During the treatment, mice were injected intramuscularly three times at four week intervals into mice at 50 μg of the pGX10, pGX10-IL-12N220L or pGX10-Ag85A DNA vectors prepared in Example 1 above. Each was then counting the number of bacteria to survive in the tissue of mice in at 18 weeks, 30 weeks, 42 weeks, i.e., two weeks after completing treatment, 14 weeks, 26 after M. tuberculosis infection (A in Fig. 3).

In addition, 30 weeks after infection, the spleen cells of the mice of each group were incubated with the culture transmission protein CF (JT Belisle, Colorado State University, Fort Collins, Colo.) Or recombinant Ag85A protein (Standardia Inc., Suwon, Korea). Afterwards, IFN- [gamma] production levels were detected in the culture. IFN- [gamma] production level determination was performed as follows. After spleen cells were taken from mice, 2 × 10 ⁵ lymphocytes were incubated for 6 days in the presence of 10 μg / ml CF or recombinant Ag85A protein at 200 μl RPMI. . Culture supernatants were collected and used for IFN-γ detection, and IFN-γ detection was determined using the mouse IFN-γ OptEIA ^™ kit (Pharmingen, San Diego, Calif.) According to manufacturer's instructions.

As a result, mice treated with INH and PZA produced IFN-γ specific for CF or Ag85A, whereas mice not treated with INH and PZA did not detect detectable levels of IFN-γ specific for CF or Ag85A. I couldn't. In control mice treated with INH and PZA without DNA immunity, IFN-γ production was induced after stimulation with CF (3ofBAnd3ofCThis indicates that the host Th1 immune response is produced by INH and PZA treatment after bacterial infection. The results are consistent with previous studies that resulted in increased IFN-γ production specific for different antigenic peptides after tuberculosis patients received chemotherapy (Dieli Fet al., Scand J Immunol,2000,52:96-102; Wilkinson RJet al., J Infect Dis,1998,178:760-768). Mice treated with control DNA (pGX10 vector) during chemotherapy showed higher levels of IFN-γ induction in response to CF and Ag85A proteins compared to control mice receiving chemotherapy only (3ofBAnd3ofCThis indicates that CpG motifs can act as inducers of Th1 immune responses in DNA vectors as previously reported (Krieg AM.Vaccine2000;19:618-622). Mice treated with IL-12N220L DNA showed a higher response to IFN-γ in response to CF than mice treated with control DNA or Ag85A DNA (3ofB, P <0.03 and p <0.04, respectively. Ag85A DNA-treated mice do not show high levels of IFN-γ induction in the event of stimulation with CF (3ofB), Induced higher Ag85A protein specific IFN-γ production than control DNA or IL-12N220L DNA-immunized mice (3ofC, P <0.01 and p <0.04, respectively. These results indicate that Ag85A-specific Th1 immune responses can be generated by Ag85A DNA immunity. IL-12N220L DNA immunity also increased the induction of Ag85-specific IFN-γ compared to control DNA immune mice (3ofC, p <0.04), indicating that the antigen-specific T-helper immune response generated after tuberculosis infection is converted to Th1-biased type by IL-12N220L DNA immunity.

Example 4 Therapeutic Effect of IL-12N220L DNA or Ag85A DNA Vaccine

<4-1> Tuberculosis Effect of DNA Vaccine in Combination with Chemotherapy

The present inventors examined the tuberculosis therapeutic effect of IL-12N220L DNA or Ag85A DNA vaccine in combination with chemotherapy through bacterial counting. Bacteria coefficient was determined from the tissue of mice at 14 weeks after the treatment with M. tuberculosis infection 30 weeks, that is, INH and PZA for 3 months completed (A and B of Figure 4 in Fig. 4).

As a result, mice treated with INH and PZA without DNA immunity showed the highest bacterial reactivity in the lung and spleen (80% and 100%, respectively). Of particular note was survival in only three mice (60%) of the lung and spleen of five mice treated with control DNA (pGX10). Tuberculosis was found, indicating that the CpG motif in the DNA vector induces a Th1 immune response, which plays an inhibitory role in M. tuberculosis reactivation.

In addition, mice treated with IL-12N220L DNA constructs in combination treatment of INH and PZA for 3 months had M in lungs and spleen of 1 (20%) and 2 mice (40%) of 5 mice, respectively. .-to Bell claw system is found (B in a and in FIG. 4 FIG. 4, Table 1) this simultaneous injection of IL-12N220L DNA appeared to be effective in inducing a Th1 immune response (Ha SJ et al., Nat Biotechnol, 2002 , 20: 381-386). Thus, IL-12N220L injection shows that it is possible to effectively attenuate immune cells against M. tuberculosis antigens and convert them into Th1 responses, thereby reducing the reactivation of M. tuberculosis in a latent infection model. Can be.

In addition, no viable M. tuberculosis was found in the lungs of all five mice treated with Ag85A DNA vaccine, and the tuberculosis bacilli were found only in the spleen of one of the five mice (20%) . A and B of FIG. 4 , Table 1 ). These results suggest that DNA vaccines expressing IL-12N220L or Ag85A in combination chemotherapy with INH and PZA inhibit the reactivation of M. tuberculosis, which is inactive in a latent infection model of tuberculosis (adjuvant). ) Can be used.

<4-2> Maintenance of Tuberculosis Treatment Effect of DNA Vaccine in Combination with Chemotherapy

To observe the continued effects of DNA vaccines on the reactivation of M. tuberculosis in a latent infection model, mice were treated with lungs at 42 weeks after Mycobacterium tuberculosis infection, 26 weeks after completion of treatment with INH and PZA for 3 months. Each bacteria was counted in the spleen.

As a result, M. tuberculosis was found in the lungs and spleens of 4 (80%) and 3 mice (60%), respectively, among 5 control mice treated with INH and PZA only, and control DNA was treated. Mice had a reactivation rate of 60% in lungs and spleen. In contrast, survival M. tuberculosis was found in the lungs and spleen of two mice (40%) out of five mice treated with IL-12N220L DNA ( Table 1 ). The results indicate that IL-12N220L DNA has a sustained effect on protective immune responses as previously reported (Ha SJ et al . Nat Biotechnol 2002; 20: 381-386).

In addition, mice treated with Ag85A DNA vaccine as shown at 30 weeks post-infection showed reactivation rates of M. tuberculosis only in lungs and spleen of 1 mouse (20%) out of 5 mice ( FIG . 4C, FIG. 4 D and Table 1 ). From these results, the reactivation rate of latent M. tuberculosis in the lungs of mice treated with IL-12N220L DNA vaccine (p <0.05, Chi-square test) and Ag85A DNA vaccine (p <0.01, Chi-square test) It can be seen that this decreases considerably. Mice treated with Ag85A DNA confer significant levels of immune defense in the spleen (p <0.05, Chi-square test), whereas the levels of protection in mice treated with IL-12N220L DNA were not statistically significant. Reactivation rates in lung and spleen were shown to be inversely related to Ag85A-specific IFN-γ induction levels, but not to CF-specific IFN-γ levels. This indicates that Ag85A-specific Th1 immunity will play a more important role in the defense of latent M. tuberculosis in therapeutic vaccines.

As discussed above, the tuberculosis DNA vaccine comprising the signal sequence of the herpes simplex virus type I glycoprotein D (gD) and the Ag85A gene of the present invention induces a humoral immune response known to be essential for the treatment of tuberculosis and a sustained immune response. Induction not only reduces the likelihood of TB recurrence, but also shortens the treatment period when used in combination with conventional chemotherapy, and can thus be useful for treating tuberculosis.

<110> POSTECH FOUNDATION          YONSEI UNIVERSITY MEDICAL CENTER <120> Therapeutic DNA vaccine for tuberculosis and pharmaceutical          composition containing the same <130> 2p-11-29B <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 60 <212> DNA <213> herpes simplex virus 7 <400> 1 atgagggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 <210> 2 <211> 1017 <212> DNA <213> Homo sapiens <400> 2 atgcagcttg ttgacagggt tcgtggcgcc gtcacgggta tgtcgcgtcg actcgtggtc 60 ggggccgtcg gcgcggccct agtgtcgggt ctggtcggcg ccgtcggtgg cacggcgacc 120 gcgggggcat tttcccggcc gggcttgccg gtggagtacc tgcaggtgcc gtcgccgtcg 180 atgggccgtg acatcaaggt ccaattccaa agtggtggtg ccaactcgcc cgccctgtac 240 ctgctcgacg gcctgcgcgc gcaggacgac ttcagcggct gggacatcaa caccccggcg 300 ttcgagtggt acgaccagtc gggcctgtcg gtggtcatgc cggtgggtgg ccagtcaagc 360 ttctactccg actggtacca gcccgcctgc ggcaaggccg gttgccagac ttacaagtgg 420 gagaccttcc tgaccagcga gctgccgggg tggctgcagg ccaacaggca cgtcaagccc 480 accggaagcg ccgtcgtcgg tctttcgatg gctgcttctt cggcgctgac gctggcgatc 540 tatcaccccc agcagttcgt ctacgcggga gcgatgtcgg gcctgttgga cccctcccag 600 gcgatgggtc ccaccctgat cggcctggcg atgggtgacg ctggcggcta caaggcctcc 660 gacatgtggg gcccgaagga ggacccggcg tggcagcgca acgacccgct gttgaacgtc 720 gggaagctga tcgccaacaa cacccgcgtc tgggtgtact gcggcaacgg caagccgtcg 780 gatctgggtg gcaacaacct gccggccaag ttcctcgagg gcttcgtgcg gaccagcaac 840 atcaagttcc aagacgccta caacgccggt ggcggccaca acggcgtgtt cgacttcccg 900 gacagcggta cgcacagctg ggagtactgg ggcgcgcagc tcaacgctat gaagcccgac 960 ctgcaacggg cactgggtgc cacgcccaac accgggcccg cgccccaggg cgcctag 1017

Claims (7)

A tuberculosis DNA vaccine comprising the signal sequence of herpes simplex virus type I glycoprotein D (gD) and the Ag85A gene. The tuberculosis DNA vaccine of claim 1, wherein the signal sequence of herpes simplex virus type I glycoprotein D (gD) is set forth in SEQ ID NO: 1. The tuberculosis DNA vaccine of claim 1, wherein the Ag85A gene is set forth in SEQ ID NO: 2. 2. The signal sequence of the herpes simplex virus type I glycoprotein D (gD) described in SEQ ID NO: 1 and the Ag85A gene described in SEQ ID NO: 2 are pGX10-Ag85A inserted into the pGX10 vector. Tuberculosis DNA vaccine (Accession No .; KCTC 10418BP). A pharmaceutical composition for treating tuberculosis, comprising the tuberculosis DNA vaccine and tuberculosis therapeutic agent of any one of claims 1 to 4 as an active ingredient. The composition of claim 5, wherein the TB treatment agent is selected from the group consisting of isoniazid (INH), pyrazineamide (PZA), rifampin, streptomycin, and ethambutol. The composition of claim 6, wherein the tuberculosis treatment agent is isoniazid or pyrazineamide.