HK1003512B - Prevention and treatment of allergic diseases by recombinant plasmid encoded allergen - Google Patents

Description

Recombinant eukaryotic vector containing allergen gene and application thereof

The present invention relates to a recombinant vector useful for preventing and treating allergic diseases, and a pharmaceutical composition comprising the same.

Allergic Diseases (AD), including allergic rhinitis, allergic asthma, and atopic dermatitis, affect about 20% of the population and are one of the major factors of illness and death. Most of the AD has recently been found to be associated with immediate hypersensitivity to inhaled allergens and to be familial-prone (this phenomenon is commonly referred to as ectopic). The etiology of AD is not clear, however, it is now found that it has many cellular and humoral deficiencies, including increased total IgE and specific IgE antibodies to multiple positive allergens. Although a number of pathogenesis concepts have been explored, the general treatment of AD is still unsatisfactory. The clinical efficacy of immunosuppressive agents such as steroids and cyclosporine in patients has improved, however, their use in children has been reduced due to their toxicity to the liver and kidney system. Even though there are currently some effective therapies with significant side effects, a subset of patients are resistant to all forms of drugs.

The main characteristic of allergic heterogeneity is the tendency to develop a persistent immunoglobulin e (ige) response to environmental antigens. IgE production is highly dependent on IL-4 and strongly inhibited by IFN-gamma. Other cytokines such as IL-5, IL-6, IL-8, IL-10 and IL-13, and cell surface molecules such as CD40 and CD23 are also involved. Recent studies with bronchoalveolar lavage fluid have shown that IgE production is regulated by suppressor T cells. Furthermore, one of the factors in the success of immunotherapy is related to the production of suppressive T cells, which can mediate the reduction of allergic reactions. There is also evidence for a defect in the function of suppressor T cells in ectopic individuals, particularly in children with allergic asthma. Recent experimental data on some animals also show that some subtypes of CD8⁺T cells may play an important role in IgE production and may inhibit allergy-induced Airway Hyperreactivity (AHR). Thus, it is possible to modulate the IgE antibody response and AHR in ectopic patients by generating antigen-specific suppressor T cells.

Previous subunit vaccination has used purified protein or viral vectors. There are, however, substantial limitations which can be overcome if the immunogenic protein is allowed to be expressed in the host cell. In this regard, genetic vaccines represent a new orientation in the development of subunit vaccines. Intramuscular injection of DNA has been shown to cause expression of the protein encoded by the DNA, as described in Wolff, J.A.et., Science 247, 1465-. Furthermore, the results show that plasmid DNA remains extrachromosomal, does not replicate, or is not inserted into the genome of the host cell, as described in Wolff j.a.etal., hum.mol.genet.1: 363-. To date, no serious inflammatory reactions or other complications have been observed at the site of inoculation. Furthermore, it is well understood that peptides derived from intracellular antigens are often presented to CD8 by Major Histocompatibility Complex (MHC) class I molecules⁺T cells, which have MHC class I molecules expressed on almost all somatic cells, and peptides derived from extracellular antigens are presented from MHC class II molecules to CD4⁺T cells, the MHC class II molecules are usually expressed by specific antigen expressing cells. Thus, the delivery of an allergen into the internal antigen processing pathway may be one that promotes antigen specificity CD8⁺T cell production, and thereby modulation of allergen-specific IgE synthesis.

In the present invention, the present inventors found that direct inoculation of a plasmid (pCMVD) encoding a group 5 allergen (Der p5) of house dust mites (Dermatophagoides pteronyssinus) into the quadriceps muscle of mice resulted in the effect of inhibiting Der p 5-specific IgE synthesis, AHR and airway inflammation in vivo upon re-challenge with Der p5, and that treatment of over-sensitized mice with different doses of pCMVD resulted in a dose-dependent therapeutic effect. The present inventors have also found that direct injection of plasmid dna (pcmvg) encoding recombinant Schistosoma japonicum (Schistosoma japonicum) protein 26(rSj26) is effective in dose-dependent modulation of rSj 26-induced allergic immune responses, including rSj 26-specific IgE synthesis and dermatitis. Therefore, the present invention infers that direct gene implantation is a method for preventing and treating allergic diseases mediated by IgE. More particularly, the present invention relates to a recombinant vector comprising a eukaryotic expression vector and an allergen gene, and a pharmaceutical composition comprising the same, which is useful for preventing and treating allergic diseases and inhibiting the production of allergen-specific IgE, and which is suitable for administration by intramuscular injection, intranasal delivery, intradermal or intratracheal delivery, etc. pCMVD and pCMVG were deposited at the institute for food industry development of New bamboo, Taiwan (FIRDI); they are designated FIRDI 940114 and 940115, deposited at 2.4.1996 and deposited at the American Type Culture Collection (ATCC) under the designation ATCC97499 and 97498, deposited at 1.4.1996.

Drawings

FIG. 1 shows the detection of the expression of Derp5 using muscle in situ cytochemical staining. Gross frozen sections of muscle taken 12 days after injection of 100. mu.g of pCMVD DNA were stained with monoclonal antibody (mAb) from Derp5(A) or Der p1(B) and optically amplified 100-fold. Sections were counterstained with hematoxylin. Similar results were obtained at all six groups of individual injection sites. Arrows indicate positively stained cells. The muscle of the control group injected with the blank vector showed no stained muscle cells.

FIG. 2 shows (A) the immune response following intramuscular injection of pCMVD. Sera from BALB/c mice that had received pCMVD injections were harvested weekly and titers against Derp5 IgG1, IgG2a and IgE were determined in the manner described in the examples. Der p 5-specific IgE was not detected in this group. Mice injected with the blank vector had no Der p 5-specific immune response. One unit of antibody corresponds to 1 microgram IgG 1/ml and 1 microgram IgG2 a/ml. (B)In vitro proliferative responses of a subpopulation of splenocytes taken from mice immunized with pCMVD. The removal of subpopulations is as described in the examples. Purified cell subsets were shown to have less than 0.5% contaminating cells by fastam analysis. CD4 from spleen of native BALB/c mice⁺Or CD8⁺Cells are added to the population to replace the removed cells. Cells were cultured with Der p5 (15. mu.g/ml) for 72 hours. Proliferation is to³Incorporation of H-thymidine was analyzed. Data shown are mean ± SD of triplicate cultures. Cells did not proliferate in response to Der p1 stimulation.

FIG. 3: (A) inhibition of the primary IgE response by pCMVD injection (intramuscularly), which is Ag specific inhibition. Mice (n ═ 6 per group) were immunized with either pCMVD or a blank vector (pCMV) as indicated. Three weeks after immunization, mice received Der p5 (10. mu.g plus aluminum hydroxide) or Der p1 (10. mu.g plus aluminum hydroxide), respectively. The data shown are mean ± SD at 21 days post challenge (n-6 per group). The titers of antigen-specific IgG2a and IgE were determined in the manner as described in the examples. P is < 0.01. (B) The inhibitory effect of adoptive transfer of splenocytes from mice immunized with pCMVD on the Der p 5-specific IgE response. CD4 used as shown by FAScam analysis^-And CD8⁺The population contained < 0.5% contaminating cells. Undigested splenocytes from mice immunized with pCMVD were used as a control group. Data shown are mean ± SD at day 21 (n-6 per group), anti-Der p5 IgG2a and IgE were determined as described in the examples. P is < 0.01. 1 unit of antibody corresponds to 1 microgram IgG2 a/ml and 100 nanograms IgE/ml.

FIG. 4 shows immunoprophylaxis of airway hyperreactivity by allergen gene transfer. Mice were injected intramuscularly with 100 micrograms of pCMV or pCMVD and sensitized with Der p5 or saline after 3 weeks. Three weeks after sensitization, mice received Der p5 or saline intake challenge. 18 hours after inhalation, pulmonary resistance was determined in the manner described in the examples. P is < 0.01.

FIG. 5 shows the inhibition of allergen-induced airway hyperreactivity by allergen gene transfer. Mice were sensitized by intraperitoneal injection of Der5 and then treated with different doses (3 microgram, 30 microgram, 100 microgram) of pCMVD plasmid DNA two weeks after sensitization. After 1 week of treatment with the synthetic plasmid, mice inhaled the allergen and measured changes in pulmonary resistance. P is < 0.01.

FIG. 6 shows the inhibition of allergen-induced airway inflammation by allergen gene transfer.

FIG. 7 is a photograph of mice treated with pCMVG (left) and pCMV (right).

FIG. 8 shows the inhibition of allergen-induced IgE synthesis by allergen gene transfer. Mice were sensitized by intraperitoneal injection with 10 microgram rSj26 plus aluminum hydroxide, and then two weeks after sensitization, treated with different doses (3 microgram, 30 microgram, 100 microgram) of pCMVD plasmid DNA. Data shown are mean ± SD at day 7 post treatment (n ═ 6 per group) and titers of anti-rSj 26 IgG2a and IgE were determined in the manner described in the examples. P is < 0.01.

The regulation of the immune response to non-pathogenic antigens is critical to the maintenance of homeostasis in immunity at the mucosal surfaces of the respiratory and gastrointestinal tracts, and failure of the control mechanisms has been considered to be a critical causative factor in allergic diseases. An important factor in this process is the selective inhibition of the Th 2-dependent IgE response to inhaled or ingested antigens, which is mediated by antigen-specific CD8⁺T cell mediated. The results of the studies conducted by the present inventors show that administration of a DNA expression vector encoding an allergen protein by intramuscular injection, intranasal delivery or intratracheal delivery or the like results in significant "split tolerance" to subsequent allergen challenge.

The present inventors have recently found that when a plasmid (pCMVD) encoding a house dust mite (serotophagoides pteronyssinus) group 5 allergen (Der p5) was inoculated directly into the mouse quadriceps muscle, a Der p5 specific T cell and IgG response was caused. This administration of plasmid DNA to mice resulted in vivo challenge with Der p5The Der p 5-specific IgE synthesis was more than 90% inhibited and AHR was also inhibited. This effect was achieved by CD8 from mice immunized with pCMVD⁺Splenocytes were transferred to other mice. The primary immune response characteristic of pCMVD-treated mice was the generation of Der p 5-specific CD8⁺T cells, which inhibit the synthesis of allergen-specific IgE and reduce AHR. The inventors have also found that direct injection of plasmid dna encoding rSj26 (pcmvg) was effective in dose-dependent fashion in reducing the rSj 26-induced allergic immune responses in mice, including dermatitis and the synthesis of rSj 26-specific IgE. Previous reports have shown that CD8 has heretofore been considered to be only a cytotoxic cell⁺T cells actually play a more active role in the regulation of immune responses. CD8⁺T cells modulate IgE production by inhibiting IgE synthesis via the inhibitory effect of IFN-gamma on B cells, and/or by affecting Th 2-like CD4 that supports IgE production⁺T cell differentiation and function. Another explanation is the possibility of CD8⁺T and B cells or CD4⁺T cells are physically exchanged and may provide inhibitory signals through homologous interactions.

Based on the above experimental results, the present invention provides a recombinant vector useful for the prevention and treatment of allergic diseases, which includes a eukaryotic expression vector and an allergen gene, such as cDNA. Prevention of allergic disease refers to the immunological prevention of allergen-induced IgE synthesis and inflammation phenomena in the target organs. Treatment of allergic disease refers to the treatment of allergen sensitized individuals to modulate IgE synthesis and inflammation in the target organs. The eukaryotic expression vector is selected from the group consisting of a vector containing a CMV promoter, a vector containing a RSV promoter or a vector containing an SV40 promoter, and is preferably pCMV. The allergen includes any environmental antigen capable of causing human allergic reaction, such as mite allergen, glutathione S-transferase of Schistosoma japonicum, house dust, animal skin debris, pollen, peanut, etc.

The present invention also provides a pharmaceutical composition for preventing and treating allergic diseases and a pharmaceutical composition for inhibiting the production of allergen-specific IgE, which comprises the recombinant vector of the present invention and a pharmaceutically acceptable carrier. The allergic diseases include, for example, allergic asthma, allergic rhinitis, atopic dermatitis and anaphylaxis. The administration of the pharmaceutical composition is preferably intramuscular injection, intranasal delivery, or intratracheal delivery. The pharmaceutically acceptable carrier may be any known carrier known in the art to be suitable for intramuscular injection, intranasal delivery, intratracheal delivery or intradermal delivery, for example, a physiologically acceptable buffer solution, physiological saline, gold beads or liposomes.

When treating a patient according to the present invention, the recombinant vector is used in a dosage of between about 0.01 and about 1.0 mg/kg body weight, which varies depending on the nature and progression of the disease to be prevented or treated and other factors, such as the age and physical condition of the patient.

Recently, research in the area of ectopy has focused almost exclusively on the treatment of established allergic diseases. In particular, treatment is focused on controlling various mediators, such as cytokines, released by end-effector cells during the immune inflammatory phase. Genetic immunization saves time and labor in generating an immune response and provides a unique method of vaccination. A novel finding of this study is that genetic immunization can lead to a "bias" in the immune response to allergens and protect animals from the attack of immune allergic asthma. The surprisingly high efficiency of plasmid DNA transfer in suppressing allergic immune responses indicates that the method of the invention provides a simple, safe, long-lasting and effective means of immunoprophylaxis and treatment of type 1 allergy.

The following examples further illustrate the invention but do not limit the scope of the invention and any alternatives and modifications known to those skilled in the art are still within the scope of the invention without departing from the spirit and intent of the invention.

Example 1A materials and methods

1. Animal(s) production

Female BALB/c 6 to 8 weeks old was used, obtained from the animal center of the university of Taiwan college of medicine (originally from Jackson laboratories, Bar Harbor, ME). In each set of experiments, mice were matched in age and gender.

2. Molecular cloning of pCMVD recombinant plasmid

Der p5 cDNA was obtained from Lin, k.l.et al, j.allergy Clin, immunol.94: 989(1994) by PCR amplification of clone WM. The 5 ' and 3 ' primers have sequences of 5 ' -AAAAAGATCTATCAT GAAATTCATC-3 '(Bgl II site at the bottom line) and 5' -ATTAAGCTTAACTTCAATCTTTTTA-3' (Hind III site at the bottom line), covering the entire Der p5 sequence. The PCR product was subcloned into the eukaryotic expression vector pCMV2 by BglII and Hind III digestion, pCMV2 was originally derived from pcDNA3(Invitrogen) and was then named pCMVD. Plasmid propagation was first carried out in SURE strains followed by Wizard^TMThe DNA purification system (Promega, Madison, Wis.) performs the purification of the plasmid according to the manufacturer's instructions. The quality and quantity of DNA were analyzed by 260 and 280nm absorbance and agarose gel electrophoresis.

3. Immunohistochemical staining of Der p5 allergen

The immunostaining is according to Histomouse-SP^TMKit (Zymed, southern san francisco, california) instructions. Briefly, frozen muscle sections (5 μm) were dried at room temperature, fixed in pure cold acetone (10 min, 4 ℃) and treated with Peroxo-Block (Zymed) for 45 sec to terminate the internal peroxidase activity. Then blocked with 10% non-immune serum for 1 hour. After blocking, the appropriate sections were incubated with Der p5mAb (from Dr. Lin. of Taiwan university) at 1. mu.g/ml for 1 hour. Der p1 mAb (4CI, mr. chapman from usa) was used as a control antibody. Mice injected with the blank vector in the same manner served as a negative control group. All incubations were carried out at 25 ℃ in a humidified chamber. After incubation, biotin-conjugated secondary antibody and streptavidin-peroxidase conjugate were added. 3-amino-9-ethylcarbazole was added to present a signal. Finally, the slide is mounted on a glass plateAnd (5) counterstaining with hematoxylin solution.

4. Determination of Der p 5-specific IgG1, IgG2a and IgE

The amounts of Der p 5-specific IgG1, IgG2a and IgE were determined by ELISA. Protein high binding plates were diluted in coating buffer (0.1M NaHCO) in 100. mu.l₃pH8.2) purified Derp5 at a concentration of 5 μ g/ml. After incubation at 4 ℃ overnight, plates were washed 3 times and blocked with 3% (w/v) BSA-PBS buffer for 2 hours at 25 ℃. For IgG, the serum used was diluted 1: 100, for IgE, the serum used was diluted 1: 10, and the measurement was performed in duplicate. After overnight incubation at 4 ℃, biotin-conjugated monoclonal rat anti-mouse IgEmAb (Phar Minigen, ca) or rat anti-mouse IgG mAb (Phar Minigen) diluted in 0.05% gelatin buffer was added and incubated for an additional 1 hour. Then, avidin-alkaline phosphatase (Sigma chemical, St. Louis, Mo.) was added, and the mixture was incubated at 25 ℃ for 1 hour, followed by washing 6 times. Phosphatase substrate p-nitrophenyl phosphate disodium salt (Sigma chemical Co.) was added. The plates were read at 405nm in a micro-test plate automatic reader (taiwan counting technology (Metertech) inc.). The readings are based on commercially available radioisotopes, and the standards are mouse anti-TNPmAb, IgG1(107.3), IgG2a (G155-178) and IgE (IgE-3) (PharMinigen).

5. Preparation of lymphocytes and cell transfer step

Purified CD4^-And CD8^-Splenocytes were obtained from the magnetically activated cell classification "MASC" (Bergisch Gladbach, germany, Miltenyi Biotec). Briefly, splenocytes taken from pCMVD immunized and control mice were co-incubated with either anti-CD 4 or anti-CD 8 monoclonal antibody coated supra-magnetic microparticles 3 weeks after immunization, 30 minutes at 4 ℃ and further co-incubated with streptavidin conjugated microparticles (Miltenyi Biotec) for 30 minutes. Labeled cells were separated from unlabeled cells by placing them in a 0.6Tesla magnetic field using a "MACS" steel wool column. Spleen cells (10) at a desired concentration⁶Acceptor) was resuspended in PBS to a final volume of 0.1 ml. To make thisThe cell suspension was injected into the tail vein of age and sex matched isogenic recipients. The receptor was then sensitized by intraperitoneal injection of 10. mu.g of Der p5 and 4 mg of aluminum hydroxide (Australian Pomber Wyeth pharmacia). Venous blood was withdrawn from the tail vein under anesthesia weekly.

6. T cell proliferation assay

The cells were purified and resuspended in complete tissue culture medium (RPMI1640) supplemented with streptomycin (100 μ g/ml), penicillin (100 units/ml), glutamic acid (5 mmol/l) and 10% heat-inactivated fetal bovine serum. Each hole has a size of 1 × 10⁵Cells were co-incubated with Der p5(15 μ g/ml) for 72 hours in 96-well flat-bottom tissue culture plates. The cells are then treated with 1. mu. Ci³H]TdR was pulsed for 18 hours, and cells were harvested using a cell harvester. Thymidine incorporation was measured in a liquid scintillation counter (Beckman, fullondon, ca).

7. Cytokine production from splenocytes from pCMVD immunized mice

IL-4 and IFN-. gamma.were measured by ELISA according to the procedure provided by the manufacturer Phar Minigen. IL-10(R & D) and TGF-. beta. (Promega) were determined using ELISA kits according to the procedures provided by the manufacturer.

8. Aerosol inhalation and analysis of pulmonary resistance

Mice were sensitized by intraperitoneal injection of 10 μ g Der p5, and 21 days after sensitization, challenged with 0.1% Der p5 diluted in PBS using an ultrasonic nebulizer. Inhalation excitation was performed in a 1 liter space attached to a De vilbis pulmonary ultrasonic nebulizer (soysseye, b., model 2512), which produces an aerosol. After 8-18 hours of exposure to aerosol, mice were anesthetized by intraperitoneal injection of Promaz and inserted into a 20-gauge tracheal tube. Changes in esophageal pressure were measured with a saline filled catheter (PE60) and a differential pressure transducer (DP 45-14 from Validyne engineering, Norsby, Calif.). The esophageal catheter was extended into the mouse esophagus until the heart effect was clearly discerned. The respiratory volume on the trachea is detected with a spirograph (Fleisch 00000, Zabona, basell, switzerland) connected to a differential pressure transducer (MP45-14, Validyne). The signals from the transducer are connected to a computer and are used by a digital electronic lung detection system (PMS, Mumed, London, UK) which calculates the lung Resistance (RL) and dynamic compliance (Ddyn) at actual time points. The experimental data are stored in telecommunication. The experimental trace or processed data is optionally spotted on a protocol printer. Acetylcholine (Ac) was administered intravenously at a starting dose of 1.25 mg/kg. The average volume per Ac dose was 10 microliters. Approximately 5 minutes apart, and only after the moderate lung pressure and volume fell within 10% of the baseline of the previous dose, and before the next dose, a 20-fold increase in Ac concentration was administered. Prior to the Ac first dose, 10 microliters of intravenous saline was administered to establish a basal value. The mean ± standard deviation of the percent change from baseline was calculated for individual animals at each Ac dose to obtain Ac dose-response curves for Der p5 sensitized or PBS Sham (Sham) sensitized groups.

9. Bronchoalveolar lavage and cell count

After measuring lung function variables, mice were lavaged with 5 x 0.5 ml aliquots of 0.9% sterile saline through tracheotomy in polyethylene tubes. The lavage solution was centrifuged (4 ℃, 500g, 10 min) and the cell pellet resuspended in 0.5 ml Hank's balanced salt solution. Add 10 μ l of cell suspension to 90 μ l of Kimura's dye and count in Neubauer chamber under light microscope to obtain total cell number. The number of differentiated cells was obtained from cytospin preparations stained with May-Grunwald dye. Cells were identified and resolved into eosinophils, lymphocytes, neutrophils and macrophages using standard morphological techniques, 500 cells were counted at 400 magnifications, and the percentage and absolute number of each cell type were calculated. B. Results

1. Immune response of plasmid pCMVD DNA immunization

To see if uptake of pCMVD DNA by muscle cells would generate an immune response, 100 μ g of pCMVD in PBS was injected into the quadriceps of female BALB/c mice. Control animals were injected with PBS or an appropriate blank vector without Der p5 gene insertion. 12 days after immunization, in situ cytochemical immunostaining was performed with anti-Der p5mAb to determine the expression of the gene transferred into muscle cells (fig. 1). In addition, the antigen-specific immune response was marked by the production of Der p 5-specific IgG1 and IgG2a antibodies, which peaked at four weeks after immunization and then gradually declined. Der p 5-specific IgE antibodies were not detected in immunized mice (fig. 2A).

2. Allergen specific T cell responses in vivo

To examine Der p 5-specific T cell responses after pCMVD injection, splenocytes were extracted from immunized mice at 3 weeks after immunization and then cultured with Der p 5. In the term of³H]Thymidine incorporation was used to analyze proliferation. At 3 weeks post-immunization, a Der p 5-specific T cell response was shown (fig. 2B). Furthermore, the proliferative response of the unfractionated splenocytes to Der p5Ag was attributed to CD4⁺Cell depletion was inhibited, but by CD8⁺Enhanced removal of cells, indicating CD4⁺Cell proliferation will be induced by CD8⁺Subpopulation inhibition.

3. Inhibition of allergen-specific IgE responses in vivo

The in vivo efficacy of pCMVD DNA injection on modulating IgE response was determined. At 3 weeks post-immunization, both vector and pCMVD treated mice were challenged with intraperitoneal injection of allergen Der p 5. Serum was analyzed for the presence of anti-Der p5IgE by ELISA three weeks after allergen challenge. In the group treated with the vector, Der p 5-specific IgE was significantly increased; in contrast, mice treated with pCMVD expressed more than 90% inhibition of Der p5 specific IgE synthesis (fig. 3A). Inhibition of IgE synthesis by pcmvdna injection was specific for Der p5, since mice treated with pCMVD produced Der 1-specific IgE when challenged with another dermatophagoides pteronyssinus allergen Der p 1. Thus, direct gene transfer can effectively suppress the synthesis of allergen-specific IgE in vivo in an allergen-specific manner.

4. Effect of T cells on inhibition of allergen-specific IgE response

Since internally expressed antigens are normally presented by MHC class 1 molecules and drive CD8⁺T cells, therefore whether Derp 5-specific IgE inhibition in vivo was determined by CD8⁺T cell induction. Non-clustered CD8⁺Removal or CD4⁺Removed splenocytes to interrupt metastasis to native recipients. The receptors were then challenged with Der p5 plus alumina adjuvant and Der p 5-specific IgE response was determined. CD4^-Both the cells and the non-clustered groups showed significant inhibition of Der p 5-specific IgE. In contrast, CD8^-The group showed no inhibitory effect, indicating CD8⁺T cells can regulate down-going IgE production (fig. 3B).

5. Cytokine production involved in inhibition of allergen-specific IgE response

CD8 was shown due to previous studies⁺T cell suppression antigen-specific antibody responses were achieved by killing Ag reactive B cells or by producing soluble factors such as IFN-. gamma., IL-4, IL-10 and TGF-. beta.and thus in vitro splenic T cells from pCMVD treated mice and cytokine production were studied. At 3 weeks after immunization with pCMVD, the spleens of the mice were removed and cultured with Der p 5. After 48 hours of incubation, the supernatants were collected and the concentrations of IFN-. gamma.IL-4, IL-10 and TGF-. beta.were determined by ELISA. Undivided splenocytes from DNA immunized mice secrete high amounts of IFN-. gamma.for specific Ag, a response to CD8⁺Removal significantly reduced, but removal of CD4⁺Cells do not have this phenomenon. At the same time, CD8⁺The population produces small amounts of IL-4. In contrast, IL-4 and IL-10 production was at CD4⁺Significant in the population. There was no difference in TGF- β production in these two groups, as shown in Table 1.

Table 1 shows the cytokine production by splenocytes from mice treated with pCMVD. Four groups of BALB/c mice (n ═ 3 per group) were immunized with plasmid pCMVD. After 3 weeks of immunization, splenocytes were cultured with recombinant Der p5(15 μ g/ml) in the manner shown. Culture supernatants were collected at 48 hours for IFN-. gamma.and IL-4, and 72 hours for TGF-. beta.and IL-10, and cytokine concentrations were measured by ELISA. Results shown are the mean of two independent experiments combined and expressed as mean ± SD.

Secretion of cytokines cells IFN-gamma IL-4 IL-10 TGF-beta

(ng/ml) (pg/ml) (unit/ml) (pg/ml) not clustered 16.5 + -2.3870.4 + -130.53.96 + -0.4362.0 + -27.5 CD4^- 11.41±1.6 198.6±46.3 1.08±0.3 219.3±21.4CD8^-0.9 + -0.5719.1 + -58.64.02 + -0.5365.2 + -30.2 culture solution 0.7 + -0.415.1 + -8.00.39 + -0.2365.1 + -20.4

Example 2A. materials and methods

1. Animal(s) production

Female BALB/c 6 to 8 weeks old, obtained from the animal center of the university of Taiwan medical school (originally from Jackson laboratories, Bar Harbor, ME), was used. In each experiment, mice of the same sex and age were selected.

2. Molecular cloning of pCMVG recombinant plasmid

rSj26 cDNA was obtained from pGEX2 by PCR amplification. The 5 ' and 3 ' primer sequences are respectively 5 ' -TAACAGATCTATGTCCCCTATACTAGG-3 '(Bgl II site at the bottom line) and 5' -TAATAAGCTTTGGAGGATGGTC-3' (Hind III site at the bottom line), encompassing the entire rSj26 sequence. This PCR product was digested with Bgl II and Hind III and cloned into the eukaryotic expression vector pCMV2, pCMV2 was originally derived from pcDNA3(Invitrogen) and was named pCMVG. Propagation of the plasmid was initially carried out in the SURE strain and then with Wizard^TMDNA purification System (Promega, Madison, Weizhou)Department) according to the manufacturer's instructions for plasmid purification. The quality and quantity of DNA were analyzed by 260 and 280nm absorbance and agarose gel electrophoresis.

3. Study procedure

Initially mice were sensitized by intraperitoneal injection with 10 microgram rSj26 and 4 mg of aluminium hydroxide (australian camper Wyeth pharmacy). 14 days after sensitization, 0.1 mg of PromAce was injected intraperitoneally^R(Ayerst laboratory, New York, N.Y.) to anaesthetize the mice. The skin was cut 10 cm so that the underlying muscles could be seen directly. The needle was inserted 0.2 cm deep into the quadriceps muscle and then 100, 30 or 3 micrograms of pCMVG or pCMV, respectively, in 0.1 ml PBS was injected into mice using a 27-gauge needle attached to a 1 ml syringe. At 7 days after gene transfer, the mice received a further challenge of 10 μ g rSj 26. At 7 days after challenge, mice were sacrificed for skin biopsy and blood sampling.

4. Purification of recombinant Sj26

Recombinant Sj26 was purified from E.coli containing the plasmid (pSj26) synthesized in accordance with leader rSj 26. Coli was grown overnight in Luria medium containing 100. mu.g of penicillin at 37 ℃ with vigorous stirring. IPTG was added to 0.1mM and incubated for an additional 3 hours. The cell pellet was washed by centrifugation at 10000g for 30 min at room temperature, and resuspended in 10mM Tris-HCl, 150mM NaCl (TBS, pH7.5) containing 5mM EDAT. Cells were broken with 0.1mM glass beads using a Braun homogenizer (b.braun, germany) in the presence of 3TIU aprotinin (Sigma), 0.5mM PMSF (Sigma) and 20 μ g/ml dnase (boehringer). Triton X-100 (1%) was added to the bacterial lysate, pre-clarified by centrifugation (40000rpm, 20 min), and the supernatant of the lysate was passed through a glutathione agar column (Sigma). After extensive washing with TBS buffer, rSj26 protein was eluted with 5mM diluted reduced glutathione in 50mM Tris-HCl (pH 8.0).

5. Determination of rSj 26-specific IgE and IgG titers in serum

rSj26 amounts of specific IgE, IgG1 and IgG2a in ELISAAnd (4) measuring. Costar high binding plates were coated with 100. mu.l of purified rSj26 or Der p5 diluted in coating buffer (0.1M NaHCO)₃pH8.2) to a concentration of 5. mu.g/ml. After incubation at 4 ℃ overnight, plates were washed 3 times at room temperature and blocked with 3% (w/v) BSA-PBS buffer for 2 hours. Serum samples were diluted in gelatin buffer 1: 100 for IgG and 1: 10 for IgE and added to the plate in duplicate. When determining specific IgE in serum, a commercially available standard (PharMinigen) is used and the reading is referenced to this standard. Serum samples and standards were incubated overnight at 4 ℃. Biotin conjugated monoclonal rat anti-mouse IgG Mo Ab (PharMinigen) diluted in 0.05% gelatin buffer was added and incubated for an additional 1 hour. Then, avidin-alkaline phosphatase (Sigma) (1: 1000) was added and incubated at room temperature for 1 hour, followed by washing 6 times. Phosphatase substrate p-nitrophenyl phosphate disodium salt (Sigma) was added for color development. The plate data were read at 405nm in a micro test disc automatic reader (Metertech). The readings were referenced to standard sera pooled from 4 mice initially injected intraperitoneally with 10 micrograms rSj26 or Der p5 plus aluminum hydroxide and at the same dose for 21 days. The standard serum was calculated as 100ELISA units/ml.

6. Prevention of allergen-induced AHR by immunization with allergen Gene transfer

Because of the efficacy and specificity of pCMVD to inhibit Der p 5-specific IgE synthesis, the inventors tested whether vaccination of pCMVD into mice could inhibit allergen-induced AHR. Lung resistance (R) was determined after Der p5 aerosol challenge with an ultrasonic nebulizer_L). In pCMV immunized mice, Der p5 sensitized mice showed significantly reduced acetylcholine doses in PC100 (14.6 ± 0.5) relative to pCMVD immunized mice (31.9 ±.2) and sham (sham) sensitized mice (30.2 ± 1.3) (fig. 4). From these results, it was found that direct gene transfer can inhibit not only allergen-induced IgE synthesis but also allergen-induced AHR.

7. Inhibition of allergen-induced AHR by direct allergen gene transfer

Since most individuals were allergen sensitized prior to treatment, the inventors investigated whether direct gene transfer could modulate AHR in allergen sensitized mice. Mice were initially sensitized intraperitoneally with 10 micrograms of Der p5 plus aluminum hydroxide and after 2 weeks of sensitization were treated with different doses of pCMVD plasmid DMA. One week after allergen gene transfer, mice received inhalation challenge and pulmonary function testing. Allergen gene transfer showed significant effects on AHR compared to sham treated mice. Thus, direct allergen gene transfer can modulate allergen-induced AHR reduction (figure 5).

8. Inhibition of allergen-induced airway inflammation by direct allergen gene transfer

In addition to determining lung function, the inventors performed bronchoalveolar lavage to study the pathogenic process of allergen-induced AHR, and to determine the effect of allergen gene transfer on airway cell infiltration. After determining lung function, mice received bronchoalveolar lavage. Neutrophils were significantly reduced in mice treated with pCMVD compared to mice treated with sham (fig. 6).

9. Histological evaluation

Each biopsy was fixed in 4% formaldehyde and then embedded in paraffin. 5-micron sections were cut out and stained by hematoxylin-eosin (eosin) staining. B. Results

1. Reduction of rSj 26-specific IgE by allergen Gene transfer

To investigate whether direct injection of rSj26 gene could modulate rSj 26-specific IgE synthesis in vivo, different doses of the pCMVG plasmid, ranging from 3 micrograms to 100 micrograms, were injected into rSj 26-sensitized mice. rSj 26-specific IgE was significantly reduced in pCMVG-treated mice. In contrast, rSj 26-specific IgG2a was not significantly different. In addition, the pCMVGDNA injection can inhibit the synthesis of rSj26 specific IgE and has specificity. Since the pCMVG treated rats produced Der p5 specific IgE when challenged with Derp 5. Therefore, direct gene transfer can effectively inhibit the synthesis of allergen-specific IgE in vivo (fig. 8).

2. Reduction of inflammatory response in skin

To investigate the efficacy of allergen gene transfer in the treatment of dermatitis, pCMVG plasmid DNA was injected into rSj26 sensitized mice. Skin tissue examination was performed on all mice 7 days after challenge injection. Histological evaluation showed significant inflammatory cell infiltration in pCMV (empty vector) -treated mice, whereas no cell infiltration was evident in pCMV-treated mice. The pCMVG treated mice also showed smooth skin, while the pCMVG treated mice showed erythema and thin skin (FIG. 7). Therefore, direct injection of the pCMVG plasmid can modulate the inflammatory phenomenon of the skin.

Claims (11)

1. A recombinant plasmid comprises a eukaryotic expression vector and an allergen gene, wherein the allergen is a Dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus) allergen and glutathione S-transferase of Schistosoma japonicum (Schistosoma japonicum).

2. The recombinant plasmid of claim 1, wherein the eukaryotic expression vector is selected from the group consisting of a vector containing a CMV promoter, a vector containing an RSV promoter, or a vector containing an SV40 promoter.

3. The recombinant plasmid according to claim 1, which is useful for the prevention and treatment of allergic asthma and atopic dermatitis.

4. The recombinant plasmid of claim 1, which is useful for inhibiting the production of allergen-specific IgE.

5. A pharmaceutical composition useful for the prevention and treatment of allergic asthma and atopic dermatitis, comprising the recombinant plasmid according to claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, which is administered by intramuscular injection, intranasal delivery, intratracheal delivery, or intradermal delivery.

7. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier is physiological saline, gold beads or liposomes

8. A pharmaceutical composition useful for inhibiting the production of allergen-specific IgE, comprising the recombinant vector of claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, which is used for the prevention and treatment of allergic asthma and atopic dermatitis.

10. The pharmaceutical composition of claim 8, which is administered by intramuscular injection, intranasal delivery, intratracheal delivery, or intradermal delivery.

11. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier is physiological saline, gold beads or liposomes.