JP5145550B2 - Pharmaceutical composition using parainfluenza type 2 virus - Google Patents

Description

  The present invention relates to a pharmaceutical composition using parainfluenza type 2 virus (PIV2).

PIV2 is a virus belonging to the Paramyxoviridae family. The genome is a single negative-strand RNA of about 15000 bases, to which a nucleocapsid protein (N) binds to form a helical symmetric nucleoprotein complex (nucleocapsid, RNP). Of the proteins encoded by the viral genome, the M protein interacts with the cytoplasmic domain of the viral glycoprotein, the envelope lipid bilayer and RNP, and is important for budding of viral particles. A viral genome from which M protein has been deleted by genetic recombination infects cells and expresses a predetermined viral protein, but cannot germinate. The present inventor has found that PIV2 lacking the M protein can be used as a vector for expressing a foreign protein in a target cell (Non-patent Document 1).
On the other hand, the present inventor has found that an α-antigen derived from acid-fast bacteria is effective for the prevention and treatment of allergic diseases (Patent Document 1).
International Publication No. 2002/066055 52nd Annual Meeting of the Japanese Society for Virology, Characterization of PIV2 with M Protein-deficient Parainfluenza Type 2 Virus (PIV2) and Mouse IL-4, November 21, 2004

In Patent Document 1, an α antigen was constructed as an expression vector pcDNA-α-K in which the α antigen was incorporated downstream of the CMV promoter and the TPA signal peptide, and was verified in an atopic dermatitis mouse model. However, in order to use the α antigen more effectively, a vector having a high expression level has been demanded. Here, although the expression level of PIV2 is large as a viral vector, there are many unknown points as to what kind of protein it can be applied to since there are few application examples.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a pharmaceutical composition and the like that can be used for the treatment and prevention of allergic diseases in which α antigen and the like are incorporated into PIV2. .

As a result of intensive studies, the present inventors have found that an expression vector incorporating an α antigen into PIV2 deficient in M protein is effective for allergic diseases by expressing the α antigen at a predetermined site, Basically, the present invention has been completed.
The present inventor has incorporated a mycobacterial-derived α antigen gene into PIV2 and administers it to the respiratory tract, so that a specific antibody can be detected not only in the respiratory mucosa but also in other mucous membranes (eg, mucosa of the digestive tract) It was found to be expressed. The fact that α antigen gene or α antigen protein can improve the immune state predominantly Th2-type cytokines, and can suppress and improve various symptoms of allergic diseases, is widely effective in the prevention or treatment of allergic diseases. Since it is clarified by the inventors' investigation (Patent Document 1), the present invention uses the α antigen more effectively.

Thus, the pharmaceutical composition for prevention or treatment of allergic diseases according to the first invention provides an α-antigen derived from mycobacteria, analogs thereof, and mutants thereof having the same function (hereinafter referred to as “α A gene encoding "antigen etc.") is incorporated into PIV2 lacking the M protein.
In addition, the method for preventing or treating allergic diseases according to the second invention comprises a deletion of M protein in genes encoding mycobacterial α-antigens, analogs thereof, and variants having functions similar to those. It is characterized by being incorporated into a PIV2 produced and administered to mammals including humans.
The α antigen is preferably derived from Mycobacterium kansasii. Moreover, it is preferable that the analog of alpha antigen is antigen 85 complex constituent protein 85A or antigen 85 complex constituent protein 85C.
The allergic disease is preferably atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, or ulcerative colitis.

  According to the present invention, when an expression vector incorporating a gene encoding an α antigen or the like into PIV2 deficient in M protein is administered to an allergic disease patient, the allergic disease is improved and a very effective effect is exhibited. can do.

  Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented. Further, the technical scope of the present invention extends to an equivalent range.

  PIV2 used in the present invention has very little pathogenicity and a large amount of protein expression. For this reason, it is not impossible to prepare and use PIV2 into which a gene encoding an α antigen or the like, which is a foreign protein, is directly incorporated. However, by deleting the M protein of PIV2, infection and protein expression are carried out in the same manner, but it becomes a safer PIV2 vector that does not produce the next generation PIV2 because it cannot germinate. For this reason, in the present invention, a gene encoding an α antigen or the like is incorporated into PIV2 lacking the M protein. The α antigen or the like may be incorporated at any position in the PIV2 genome, but it is preferable to incorporate it at the 5 ′ end side of the antisense virus genome since the expression level of the foreign protein increases. Antisense PIV2 genome is a single-stranded RNA, from 5 ′ end to 3 ′ end, leader (Leader: promoter sequence), NP, V / P, M, F, HN, L, trailer. Has the structure. For this reason, the gene encoding the α antigen or the like is preferably located immediately after the leader (promoter sequence) or at a position close to the 5 ′ end such as between NP and V / P.

Examples of the deletion of the M protein include a method of removing all or part of the gene portion encoding the M protein, or a method of incorporating a stop codon somewhere in the gene encoding the M protein.
In the present invention, an allergic disease is a disease that reacts sensitively to environmental antigens / self-antigens, etc., to which normal persons do not react, and causes destruction and damage of each organ by its own immune system. Specific examples include atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, ulcerative colitis and the like, but the application of the present invention is not limited to these specific diseases.

  In the present invention, the α antigen is one of proteins universally present in acid-fast bacteria and has been identified as the antigen 85 complex-constituting protein 85B, which is one of the antigen 85 complexes. This antigen 85 complex includes an antigen 85 complex constituting protein 85A (Infect. Immun. 57: 3123-3130, 1989) and an antigen 85 complex constituting protein 85B (J. Bacteriol. 170: 3847) having a molecular weight of about 30 to 32 kd. -3854, 1988), and antigen 85 complex component protein 85C (Infect. Immun. 59: 3205-3212, 1991), these are the major secreted proteins of mycobacteria. These secreted proteins are highly homologous among species of the same genera such as Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium kansasii, and show cross-reactivity to monoclonal antibodies (Microbiol. Rev. 56: 648-661, 1992). Examples of analogs of the α antigen (antigen 85 complex constituting protein 85B) include antigen 85 complex constituting protein 85A, antigen 85 complex constituting protein 85C, and the like.

  In the present invention, the gene encoding the mycobacterial α-antigen or an analog thereof is an α-antigen protein or an analog of an α-antigen protein such as an antigen 85 complex-constituting protein 85A or antigen 85 complex-constituting protein 85C. It refers to a gene that can be expressed. Specifically, a gene in the form of an expression vector containing a gene encoding an α antigen or an analog thereof can be mentioned. Examples of the gene encoding the α antigen include Mycobacterium kansasii (Infect. Commun. 196: 1466-1473, 1993), Mycobacterium leprae (Mol. Microbiol. 6: 153-163, 1992), and the like. Any of these genes can be used in the present invention. Among these, as a gene encoding an α antigen of Mycobacterium kansasii, DNA having a nucleotide sequence from 390th to 1244th shown in SEQ ID NO: 1 can be mentioned. In addition to this DNA, one or more (preferably several) amino acid residues are substituted for the amino acid sequence of the mutant DNA that hybridizes with this DNA under stringent conditions and the protein encoded by this DNA. Alternatively, a mutant DNA encoding a protein consisting of a deleted and / or added amino acid sequence and encoding a protein having the same function as the α antigen derived from Mycobacterium kansasii can also be used. The function similar to the α antigen derived from Mycobacterium kansasii has the same effect of preventing or treating allergic diseases.

  Specific methods for obtaining mutant DNA include, for example, the following methods. That is, 50% formamide, 4 × Denhardt, 5 × SSPE (SSPE solution: EDTA phosphate sodium salt (SSPE), 1 × Denhardt: 0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.2% SDS, 100 μg / ml ssDNA, and 12.5 ng of a probe (12.5 ng of a cDNA fragment having the base sequence from the 390th to 1244th positions shown in SEQ ID NO: 1 were obtained using BcaBest DNA Labeling Kit (TaKaRa) [α- 32P] in the presence of dCTP (Amersham)), and colony hybridization was performed at 45 ° C. for 14 to 16 hours, and the filter was washed with 1 × SSPE, 0.5% SDS solution at 45 ° C. for 30 minutes. After washing in 0.1 × SSPE, 0.5% SDS solution at 55 ° C. for 1 hour, and finally in 0.1 × SSPE, 0.5% SDS solution at 65 ° C. for 1 hour, after removing the background, X Mutant DNA can be obtained by exposing to line film (Fuji) at −80 ° C. for 72 hours, locating and isolating the corresponding colonies. The amino acid sequences encoded by these variants usually have a homology of 60% or more, preferably 75% or more, with respect to the amino acid sequence of the α antigen. Similarly, in the case of a gene encoding an α-antigen derived from acid-fast bacteria other than Mycobacterium kansasii, these mutants may also be used.

  Examples of the gene encoding the antigen 85 complex constituting protein 85A, which is an analog of the α antigen, include genes derived from acid-fast bacteria similar to the various acid-fast bacteria for the α antigen gene described above. More specifically, DNA encoding the antigen 85 complex-constituting protein 85A derived from Mycobacterium tuberculosis (Infect.Immun. 57: 31323-3130, 1989) can be mentioned. Similarly, the gene encoding the antigen 85 complex constituting protein 85C includes genes derived from various acid-fast bacteria, and more specifically, DNA encoding the antigen 85 complex constituting protein 85C derived from Mycobacterium tuberculosis. (Infect.Immun.59: 3205-3212, 1991). In the same manner as described above, these DNAs may be one or more (preferably several) of DNAs that hybridize with these DNAs under stringent conditions, or the amino acid sequences of proteins encoded by these DNAs. A DNA encoding a protein comprising an amino acid sequence in which an amino acid residue is substituted, deleted and / or added, and having a function similar to that of antigen 85 complex constituting protein 85A or antigen 85 complex constituting protein 85C Encoding variants can be used.

  The above DNA is subjected to RT-PCR reaction on mRNA derived from mycobacteria using an appropriate DNA portion as a PCR primer based on the sequence information described in the above literature, the sequence information such as Genbank, etc. Can be cloned. It can also be chemically synthesized based on amino acid sequence information. The mutant of DNA can be easily obtained by, for example, site-directed mutagenesis, PCR, or ordinary hybridization.

  In the present invention, an α-antigen protein derived from acid-fast bacteria, an antigen 85 complex-constituting protein 85A or an antigen 85 complex-constituting protein 85C per se, or a mutant protein thereof is used for the prevention or treatment of allergic diseases. It can also be used. Examples of such an α antigen include a protein encoded by the gene encoding the α antigen described above. Specifically, for example, an α antigen encoded by DNA having a base sequence derived from Mycobacterium kansasii shown in SEQ ID NO: 1 and having an amino acid sequence shown in SEQ ID NO: 2 can be mentioned. In addition to the α antigen having this amino acid sequence, a protein comprising an amino acid sequence in which one or more (preferably several) amino acid residues are substituted, deleted, and / or added to the amino acid sequence of SEQ ID NO: 2. Thus, it may be a mutant protein having the same function as the α antigen. In the case of α-antigens derived from acid-fast bacteria other than Mycobacterium kansasii, amino acids in which one or more (preferably several) amino acid residues are similarly substituted, deleted, and / or added to their amino acid sequences. It may be a mutant protein having a sequence and a function similar to that of the α antigen. In addition, regarding the antigen 85 complex constituting protein 85A or the antigen 85 complex constituting protein 85C, the antigen 85 complex constituting protein 85A derived from Mycobacterium tuberculosis (Infect.Immun. 57: 31323-3130, 1989), the antigen derived from Mycobacterium tuberculosis 85 complex constituent protein 85C (Infect.Immun.59: 3205-3212,1991) etc. are mentioned. These α antigen protein analogs may also be the same mutant as the α antigen protein mutant.

Such a protein can be produced by a recombinant DNA method using a gene encoding the protein, or can be produced by chemical synthesis. Alternatively, it can also be obtained by culturing an acid-fast bacterium such as Mycobacterium kansasii in an appropriate medium and purifying it from the culture by a known purification method (Scan. J. Immunol. 43: 202-209, 1996; J. Biol. Bacteriol.170: 3847-3854, 1988; Hiroshima J. Med.Sci.32: 1-8, 1983).
In the present invention, when using a gene encoding an alpha acid derived from an acid-fast bacterium, an analog thereof, or a mutant thereof for the prevention or treatment of allergic diseases, specifically, an α derived from an acid-fast bacterium is used. It is used in the form of PIV2, which contains the gene encoding the antigen, its analogs or variants thereof. Recombinant PIV2 can use the PIV2 genome from which the M protein has been deleted in advance. A PIV2 vector expressing an α antigen or the like is constructed by incorporating a gene encoding an α antigen or the like into the M protein-deficient PIV2 genome.

  This expression vector can usually be administered to mammals including humans in the form of sprays, oral agents, transmucosal agents, injections and the like. Each agent can be prepared by a conventional method. For example, it can be prepared by dissolving in an appropriate solvent (buffer solution such as PBS, physiological saline, etc.), then sterilizing by filtration with a filter or the like as necessary, and then filling in an aseptic container. A conventional carrier or the like may be added to each agent as necessary. In order to use the expression vector thus obtained for the treatment of allergic diseases, it can be administered by a usual method.

An expression vector containing a gene encoding an α antigen or the like is usually administered to the skin, mucous membrane, muscle, abdominal cavity and the like of mammals including humans. The dose may vary depending on the administration form, administration method, subject patient, disease type, etc., but generally, the expression vector is about 1 × 10 ^{6 to} 1 × ^{10 10} viruses, preferably about 1 × 10 ^{7 to} 1 × 10 ⁹ It is a virus, and is preferably administered once a day for a few months, a total of 2-3 times.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these Examples.

Example 1 <Construction of antisense rPIV2 genome lacking M protein>
FIG. 1 shows an outline of an antisense rPIV2 genome in which a stop codon is incorporated at a predetermined position of a gene encoding M protein. FIG. 1 shows two types of M: Δ119 (A protein at position 259 of the M protein gene is TAG) and Δ289 (ATG at position 89 of the M protein gene is TAG, and AAG at position 259 is TAG). A state of a plasmid vector in which a protein-deficient PIV2 (rPIV2 (Δ119) and rPIV2 (Δ289), respectively) antisense rPIV2 genome is incorporated as a cDNA downstream of the T7 promoter is shown. In order to construct PIV2 deficient in M protein, a genetic technique (for example, PCR method) known to those skilled in the art can be used.

Example 2 <Preparation Method of rPIV2>
Next, a method for recovering virus particles from a plasmid containing an antisense rPIV2 genomic cDNA inserted downstream of the T7 promoter will be described. FIG. 2 shows an outline of the method. Antisense rPIV2 genomes (rPIV2, rPIV2 (Δ289), rPIV2 (Δ119)) constructed as shown in FIG. 1 were transfected into cells expressing T7 RNA polymerase (for example, BSR-T7 / 5). At this time, three types of expression vectors PIV2-NP, PIV2-P, and PIV2-L, which are vectors that express the PIV2 polymerase unit (ie, NP protein, P protein, and L protein), were transfected together. For DNA transfection, methods well known to those skilled in the art can be used (in this embodiment, lipofectamine was used).

When co-culture with Vero cells was performed 5 to 7 times every 48 hours, a cytopathic effect (CPE) was confirmed with an efficiency of 90% or more. At this time, in rPIV2, a large amount of recombinant virus particles were observed in the supernatant, but in rPIV2 (Δ289) and rPIV2 (Δ119), almost no recombinant virus particles were confirmed. This indicates that PIV2 does not germinate when the M protein is deleted.
Therefore, instead of Vero cells, co-culture was performed using Cos cells expressing PIV2-M (Cos cells transfected with PIV2-M, which is a vector expressing M protein). As a result, a large amount of recombinant virus particles were also observed in the supernatant for rPIV2 (Δ289) and rPIV2 (Δ119).
In the above description, the method for preparing rPIV2 using the T7 promoter has been described. However, according to the present invention, any rPIV2 prepared using any vector (phage vector, plasmid vector, etc.) can be used. .

Example 3 <Construction of rPIV2 containing gene encoding α antigen>
M protein deficiency in which the α antigen gene is incorporated by inserting the α antigen gene (α-K) of Mycobacterium kansasii consisting of the 390th to 1244th base sequences shown in SEQ ID NO: 1 into the NotI site of M protein deficient rPIV2 rPIV2 (hereinafter referred to as “rPIV2-αK” or “PIV2Ag85B”) was constructed (see FIG. 3). The NotI site was incorporated immediately after the leader sequence of the rPIV2 genome and is located near the 5 ′ end of the antisense rPIV2 genome. As a control, rPIV2 (hereinafter referred to as “rPIV2-EGFP”) in which an EGFP (fluorescent protein derived from Echinella jellyfish) gene was incorporated into the NotI site was constructed.
RPIV2-αK and rPIV2-EGFP were prepared in large quantities by the method of Example 2.

Example 4 <Confirmation of EGFP expression in hamster>
When rPIV2-EGFP was administered intranasally to hamsters (IN: 5 × 10 ⁵ virus), EGFP fluorescence was confirmed in epithelial cells of the respiratory tract and lungs. From this, it was found that rPIV2 promotes the expression of extremely high foreign genes in the respiratory tract and lungs by nasal administration.
Example 5 <Confirmation of effect on ovalbumin sensitization using mouse>
Solution of 4 groups (6 mice / group) of BALB / C mice in which 10 μg of ovalbumin (OVA) and 1 mg of Alum (aluminum hydroxide) were dissolved in 500 μl of phosphate buffer / saline (PBS). On day 1 and day 14 of the experiment, immunization was carried out by intraperitoneal administration (IP), and a 5% -OVA (PBS) solution was inhaled by aerosol for 5 days from day 21 to prepare an asthma model. Each group was treated as follows.

Group 1: PBS (IP) -PBS (IN): In preparing the above asthma model, instead of OVA, a solution of 1 mg of Alum dissolved in 500 μl of PBS per mouse was IPed. In addition, 20 μl of PBS was added to each of the experiments on the 20th day (the day before OVA sensitization).
Group 2: OVA (IP) -PBS (IN): Start of experiment-On day 20 (the day before OVA sensitization), 20 μL of PBS was administered intranasally (IN).
Group 3: OVA (IP) -PIV2 (IN): Start of experiment-On day 20 (the day before OVA sensitization), 1 × 10 ⁷ viruses / 20 μl of rPIV2 were added.
Group 4: OVA (IP) -PIV2Ag85B (IN): Start of experiment-On day 20 (the day before OVA sensitization), 1 × 10 ⁷ virus / 20 μl of rPIV2-αK was added.
For each group, on the 25th day from the start of the experiment, serum OVA-specific IgE concentration (OVA-specific IgE), total cell number in total bronchoalveolar lavage fluid (BALF), total protein concentration (Total protein) ), Interleukin 5 concentration (IL-5), and interleukin 13 concentration (IL-13). In addition, eosinophils in the lung tissue were stained (Meigemsa staining), and the number of cells was measured with a microscope.

The results are shown in FIGS. As shown in FIGS. 4 to 8, in the second group or the third group, the serum OVA-specific IgE concentration, the total number of cells in BALF, the total protein concentration, IL-5, and IL-13 concentration are as follows: Compared to the first group, there was a significant (p <0.01) increase. In addition, no significant difference was observed between the second group and the third group.
In the fourth group, a significant decrease was observed for all data compared to the second and third groups (p <0.01). Further, the total protein concentration and the IL-13 concentration were reduced to such a level that no significant difference was observed between the fourth group and the first group.
As shown in FIG. 9, infiltration of eosinophils was observed in the second group and the third group as compared with the first group. On the other hand, in the fourth group, compared with the second group and the third group, these inflammation images were clearly reduced, and almost no difference was observed from the first group.
From these results, it was revealed that PIV2Ag85B acts very prophylactically and therapeutically on allergic inflammatory reactions caused by OVA. In particular, in Patent Document 1 previously clarified by the present inventors, although a preventive effect is recognized, it was difficult to sufficiently recognize the therapeutic effect, but in this embodiment, the therapeutic effect is also sufficiently proved. It was.

Example 6 <Confirmation of immune effect of α antigen in mouse>
After rPIV2-αK was intranasally administered to mice (IN: 1 × 10 ⁷ virus), the amount of various antibodies against PIV2 in serum and feces (200 mg / ml) was measured 18 days later. The results are shown in FIGS. From the figure, it was shown that specific antibody against PIV2 increases not only in serum but also in stool by nasal administration of rPIV2-αK. This means that the α antigen expressed from rPIV2 taken nasally showed an immunological effect not only on the local site but also on other mucosa.

Thus, by administering rPIV2-αK nasally, in addition to being effective for allergic rhinitis, which is a local allergic disease, and allergic inflammatory diseases in the respiratory tract represented by asthma, It has also been shown to be effective for allergic diseases (for example, atopic dermatitis, allergic conjunctivitis, ulcerative colitis) at the site.
As described above, according to this embodiment, administration of an expression vector incorporating a gene encoding an α antigen or the like into PIV2 deficient in M protein to allergic disease patients leads to improvement of allergic diseases and is extremely effective. It was possible to demonstrate the effect.

It is a figure explaining the outline | summary of the antisense rPIV2 genome which deleted M protein. It is a figure explaining the method to collect | recover the viral particle from the plasmid vector containing the antisense rPIV2 genomic cDNA integrated downstream of T7 promoter. It is a figure which shows the structure of M protein defect | deletion antisense rPIV2 (rPIV2- (alpha) K) which integrated the alpha antigen gene. It is the figure which compared the effect by nasal administration in an asthma-like model mouse with the following parameters, the 1st group (PBS), the 2nd group (OVA), the 3rd group (PIV2), and the 4th group (PIV2-Ag85B) ) Is a graph comparing the total number of cells in BALF. It is the graph which compared the total protein density | concentration in BALF in 1st group-4th group. It is the graph which compared the OVA specific IgE density | concentration in serum in 1st group-4th group. It is the graph which compared the IL-5 density | concentration in BALF in the 1st group-the 4th group. It is the graph which compared IL-13 density | concentration in BALF in 1st group-4th group. In the 1st group-the 4th group, it is a typical photomicrograph figure when the inflammatory image of a respiratory organ is observed. It is a graph which shows the amount of IgA in the serum of the animal which administered rPIV2- (alpha) K nasally, and feces. naive is data of animals not administered with rPIV2-αK, and PIV2 / Ag85B is data of animals administered with rPIV2-αK. Of the paired data, the left side shows serum data (x100), and the right side shows fecal data (x5) (the same applies to FIGS. 11 and 12). It is a graph which shows the amount of IgG1 in the serum and the stool of an animal administered nasally with rPIV2-αK. It is a graph which shows the amount of IgG2a in the serum of the animal which administered rPIV2- (alpha) K nasally, and feces.

Claims (5)

Immediately after the leader sequence of the parainfluenza type 2 virus ( PIV2 ) gene genome deficient in the M protein, a gene encoding an alpha antigen derived from mycobacteria can be incorporated, and the gene encoding the alpha antigen can be expressed in vivo A pharmaceutical composition for the prevention or treatment of allergic diseases, which is a pharmaceutical composition for the prevention or treatment of allergic diseases. The α antigen, Mycobacterium Kanzashii (Mycobacterium kansasii) prophylactic or therapeutic pharmaceutical composition for allergic diseases according to claim 1, characterized in that is derived from. The pharmaceutical composition for preventing or treating allergic diseases according to claim 1 or 2, wherein the α antigen is antigen 85 complex constituting protein 85A or antigen 85 complex constituting protein 85C. 3. The pharmaceutical composition for prevention or treatment of allergic diseases according to claim 2, wherein the α antigen is encoded by the nucleotide sequence from the 390th to 1244th nucleotide shown in SEQ ID NO: 1. The pharmaceutical composition for prevention or treatment of allergic disease according to any one of claims 1 to 4 , wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis, or allergic conjunctivitis .