Vaccination with the DNA encoding T-cell epitopes to the house dust mite Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f were effective in the inhibition of the allergen induced IgE synthesis. Gene therapy using T-cell epitope encoding DNA is useful in combating allergic disease.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to a method of vaccination using naked DNA encoding T-cell epitopes, such as those of the house dust mite Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f), which results in the suppression of IgE production. The present invention also relates to novel combinations of mixed plasmid DNA useful in IgE suppression.

BACKGROUND OF THE INVENTION

Genetic vaccination with naked plasmid DNA provides a long standing cellular and humoral immune response and promotes a shift in the pattern of cytokines produced by the T-cells. Peptides derived from T-cell epitopes can downregulate cytokine production and prevent specific antibody formation and administration of a single dominant epitope may tolerize the response to all the T-cell determinants within that protein.

About 15% of the world population exhibit a hypersensitivity response to common aeroallergens resulting in asthma, eczema, and rhinitis. The most frequently implicated allergens are derived from the house dust mite (HDM) including Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). From the serological analysis of IgE antibodies from HDM-allergic individuals, a major component (>90%) had a humoral response that was reactive with the group 1 and group 2 allergen. Therefore, using truncated recombinant proteins and overlapping peptides based on the nucleotide sequences, it is possible to generate T-cell epitope maps for human responses to HDM-derived allergens and to allow the development of immunotherapy. However, vaccines using peptides has a substantial limitation that in the peptides are poor immunogens. Recently, studies have shown it has revealed that genetic vaccinations with naked DNA provide long-lasting cellular and humoral immune responses. Long-term persistence of plasmid DNA and foreign gene expression in muscle suggested that muscle is an attractive target tissue for gene vaccination. Many studies have revealed that gene immunization with plasmid DNA encoding whole allergens or protein antigens induced strong T helper type (Th1) immune responses in mice and rats.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method of vaccination using naked DNA encoding T-cell epitopes, such as those of the house dust mite Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f), which results in the suppression of IgE production. The present invention also provides novel combinations of mixed plasmid DNA useful in IgE suppression.

The invention also includes a composition for reducing IgE production, comprising: a pharmacologically acceptable medium and a substantially pure, immunogenic plasmid DNA encoding a T-cell eptitope. The present invention provides a method of reducing IgE production, comprising administering a composition comprising a pharmacologically acceptable medium and a substantially pure, immunogenic plasmid DNA encoding a T-cell eptitope. The present invention also provides a vaccine for reducing the severity of an allergic disease in a mammal, comprising a pharmaceutically acceptable carrier and at least one plasmid DNA that encodes a T-cell epitope from a house dust mite antigen wherein the dust mite is selected from the group consisting of the house dust mite Dermatophagoides pteronyssinus (Der p), Dermatophagoides farinae (Der f) and mixtures thereof. The present invention further provides a composition for reducing IgE production, comprising: a pharmacologically acceptable medium and a substantially pure, immunogenic plasmid DNA encoding a major HDM allergen selected from the group consisting of Der p 1, Der p 2, Der p 3, Der f 1, Der f 2, Der f3 and mixtures thereof.

EXAMPLES

Example 1

Human Epitope Vaccination

Introduction

To determine whether the vaccination with naked plasmid DNA encoding only a T-cells epitope peptide is able to suppress the allergic reaction in vivo, the mixed naked DNA plasmids encoding the five classes of human T-cell epitopes on Der p 1 and Der p 2 were used for genetic vaccination of BALB/c mice. The control mice were injected with the pcDNA 3.1 blank vector. There was a reduction in the total and Der p-specific immunoglobulin E (IgE) synthesis in the vaccinated mice compared with the control mice. In the Der p specific-IgG2a antibody response, the vaccinated mice showed more prominent responses than the control mice. Also analysis of the cytokines serum levels after immunization of Der p extract revealed that in the vaccinated mice there was an elevation in the level of interferon-.gamma., a Th1 cytokine associated with suppression of IgE production. The histologic studies showed that there was much less infiltration of inflammatory cells observed in lung tissue of the vaccinated mice than that of the control mice.

To evaluate whether the vaccination with naked plasmid DNA coding only a T-cell epitope peptide suppresses allergic reactions as effectively as the vaccination with DNA encoding whole allergen, an immune response to gene immunization with plasmid DNA encoding major T-cell epitopes in Der p 1 and 2 to challenges with whole Der p extract in mice to mimic realistic clinical setting was investigated. It was demonstrated that genetic vaccination indeed induced strong Th1 immune responses which reduced the IgE antibody production and allergic responses against Der p. Therefore, it would be ideal to develop an alternate naked DNA vaccination method which could be even safer than injecting whole segments of the allergen encoding region of either Der p1 or Der p2.

Materials and Methods

Mice

20 BALB/c mice at the age of 6-8 weeks were purchased from Jackson Laboratory (Bar Harbor, Me.) and bred at the University of Tennessee (Memphis, Tenn.). This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animal Welfare Act (7 U.S.C. et seq.); the animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee.

Plasmid Construction

Total mRNA was isolated from Der p and Der f HDM, respectively. By using murine leukemia virus reverse transcriptase and random hexanucleotide primer following the instructions of the Perkin Elmer Gene Amp RNA PCR kit (Perkin Elmer, Branchberg, N.J.), first-strand cDNA was generated from 1 .mu.g of total RNA and subjected to RT-PCR. The cDNA was used in PCR with Taq polymerase and with primers specific for human T-cell epitopes of Der p 1 and 2. These primers, which cover the mature excreted region of each genes and include EcoRI and XbaI sites for cloning, summarized in Table 1 (see Original Patent). The amplified PCR products were subcloned into pcDNA3.1 eukaryotic expression vector (Invitrogen, San Diego, Calif.) and then sequenced.

DNA Preparation and Vaccination

Each plasmid construct was prepared using Maxi prep (Qiagen, Chatsworth, Calif.). Mice were vaccinated by injection with 300 .mu.g of pcDNA3.1 blank vector in 100 .mu.l of PBS (control mice) or with 300 mg of the mixed naked DNA encoding the human T-cell epitopes of Der P 1 and 2 in 100 .mu.l of PBS (vaccination mice) three times at weekly intervals into muscle.

Immunization and Inhalation of Allergen to Mice

The Der p-induced sensitivity in a mouse model was performed as described, Der p was emulsified with an equal volume of complete Freund's adjuvant (CFA) for immunization. Three weeks after last vaccination, mice were sensitized by injecting subcutaneously at the base of the tail with 100 .mu.g of Der p extract in CFA. The mice were also given an intraperitoneal dose of 300 ng of purified pertussis toxin at 24 and 72 hours after first immunization. Seven days later, the mice were boosted again with the same amount of antigen in incomplete Freund's adjuvant. Mice received intranasally by inhalation intranasal with 10 .mu.g of Der p extract six times at weekly intervals from boost.

Determination of Total and Der p-Specific IgE

The bloods from the 7 mice in two groups were collected three times on week 0 (first immunization), 3, and 6. The total IgE level was determined by ELISA as follows. One hundred microliters of anti-mouse IgE capture mAb (clone R35-72; Pharmingen, San Diego, Calif.) were added in each well to plate and incubated overnight at 4.degree. C. After washing, two hundred microliters of 10% fetal calf serum were incubated at room temperature for 30 min. The plates were washed five times with washing buffer and incubated with the diluted mouse serum overnight at 4.degree. C., followed by the addition of one hundred microliters of HRP-conjugated anti-mouse IgE detection mAb (clone R35-1 18; Pharmingen, San Diego, Calif.) overnight at 4.degree. C. The plates were washed five times before adding citric acid-phosphate buffer (pH 5.0) containing 0.15 mg/ml of O-phenylenediamine (Sigma, St. Louis, Mo.). The color was developed at room temperature, and the reaction was stopped by 2.5 M sulfuric acid. The color was measured at 492 nm (Bio-Rad, Richmond, Calif.). The purified mouse anti-IgE antibody (Pharmigen, San Diego, Calif.) was used for total IgE standard. In the measure of the Der p specific IgE, the plate were coated with 25 .mu.g/ml of Der p in 0.1 M carbonate buffer (pH 9.6) and serum samples were diluted fivefold in 10% FCS. The other procedures were the same as the measurement of Der p-specific IgE. The levels of Der p-specific IgE were referenced to the standard serum pooled from six mice that were immunized with 100 .mu.g of Der p twice and inhaled with 10 .mu.g of antigen six times. The standard serum was calculated as 100 ELISA units/ml.

Determination of Der p Specific IgG, IgG1, and IgG2a

The Der P specific IgG, IgG1, and IgG2a were determined by ELISA as follows. Purified antigens (5 .mu.g/ml) were coated onto the assay plate and incubated overnight at 4.degree. C. The other procedures were the same as the measurement of Der p-specific IgE.

Cytokines Serum Levels in Balb/c Mice after Immunization with Der p

Blood samples from the 7 mice in two groups were collected two times on week 0 (first immunization), and 2 weeks. The levels of IFN-.gamma. and IL-4 were measured using the antibody pairs purchased from PharMingen, according to the manufacturer's instructions.

Histological Examination of Lung Tissue

Mice were anesthetized with a mixture of ketalar (35 mg/ml), rompun (0.6%/ml) and atropine (0.1 mg/ml), of which 0.2 ml was injected intramuscularly. The vascular bed of the lungs was perfused with 0.01 M Phosphate-buffered saline (PBS) and then with 4% paraformaldehyde 0.1 M PBS buffers. Whole lungs were taken out and were stored in 4% paraformaldehyde for 24 h at 4.degree. C. After fixation, these tissues were dehydrated and embedded in paraffin. Frozen sections are cut at 6 .mu.m in thickness were stained by hematoxylin and eosin. After coding, the sections were evaluated by two observers using light microscopy. The amount of mononuclear cells per section was scored using the method described by Hessel et al. This scoring method discriminates between the presence of mononuclear cells around blood vessels (score 0-3), and around bronchioli (score 0-3), and the number of patchy cellular infiltrates (score 0-3). Histological scores were analyzed using non-parametric Mann-Whitney U test. At least five mice were examined.

Lymph Node Cell Proliferation

The proliferation assay was performed as described. Briefly, 10 days after immunization, lymph nodes were removed aseptically, and single-cell suspension was prepared. The cells (2.times.10.sup.5 cells per well) were cultured with the serial dilution of Der p (range, 0.01-10 .mu.g/ml). Cultures were set up in 200 .mu.l RPMI1640 supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, Utah), 1 mmol/L sodium pyruvate, 100 .mu.g/ml penicillin, 100 .mu.g/ml streptomycin, 2 mmol/L glutamine, 5.times.10.sup.-5 mol/L 2-mercaptoethanol, 20 mmol/L HEPES (pH 7.4), and 50.times. nonessential amino acids. After 72 hours culture, 1 .mu.Ci of [.sup.3H] thymidine (Du Pont, Wilmington, Del.) was added to each well. Eighteen hours later, cells were harvested, and measured by liquid scintillation counting. Values were expressed in counts per minute as follows: Counts per minute with antigen-Counts per minute without antigen. Each sample was run in triplicate. RPMI medium 1640, sodium pyruvate, penicillin, streptomycin, glutamine, HEPES, and 50.times. nonessential amino acids were purchased from Irvine Scientific (Santa Ana, Calif.), and 2-mercaptoethanol was purchased from Sigma Chemical Co. (St. Louis, Mo.).

Statistical Analysis

The immunoglobulin response data was analyzed by Student's paired t test for comparisons between control and experimental mice. Histological grades were analyzed using a non-parametric Mann-Whitney U test. Data was expressed as mean .+-.SD. A P value <0.05 was considered significant.

Results

Suppression of total and Der p-specific IgE antibody production by gene vaccination.

To determine the effect of vaccination with DNA encoding T-cell epitopes, we examined total and Der p specific IgE antibody levels by ELISA (FIGS. 1A and 1B, see Original Patent). The gene vaccination with the human T cell epitopes of Der p 1 and 2 showed about 50% inhibition of Der p-specific IgE and more than 50% inhibition of total IgE as compared with the control mice at week 6. Thus, genetic vaccination could inhibit an in vivo allergen-specific IgE synthesis efficiently. To study the effects of DNA vaccination on B cell immunity, we measured Der p specific serum antibodies. The increase in production of Der p-specific IgG2a antibodies in the vaccination mice was greater than that in the control mice after 3 weeks although Der p specific IgG responses were similar between the two groups (FIGS. 1C and 1D, see Original Patent). But in the Der p-specific IgG1 response, control mice showed more prominent production than vaccination mice (FIG. 1E, see Original Patent).

IFN-.gamma. and IL-4 Serum Levels in Balb/c Mice after Immunization with Der p Extract

To determine whether the Th1 or Th2 cytokines were produced in response to genetic vaccination with the human T-cell epitope genes, we measured IL-4 and IFN-.gamma. serum levels. The IFN-.gamma. serum level in vaccination mice (648.29.+-.166.78 pg/ml) was observed to be higher than in control mice (undetectable) at 2 weeks after immunization of Der p extract (See FIG. 1F, see Original Patent). In parallel, the IL-4 serum levels were detected contrary to the result of IFN-.gamma. (control mice 23.63.+-.3.66 pg/ml versus vaccination mice undetectable) (see FIG. 1G, see Original Patent). Our results suggested that the genetic immunization with the plasmid DNA encoding T-cell epitopes might also induce a Th2 to Th1 cytokine shift.

Lymph Node Cells Proliferation

To determine whether the protective effect of gene vaccination was due to the deletion of T-cells or the induction of unresponsiveness, we examined lymph node cell proliferation in response to different concentrations of Der p. As seen in FIG. 1H (see Original Patent), lymph node cells from the vaccinated mice showed a linear stimulation when concentration of Der p ranged from 0.01 .mu.g/ml to 10 .mu.g/ml, as did the lymph node cells from the control mice.

Histological Examination of Lung Tissue

To examine whether the genetic vaccination affected cellular response of lung or not, we stained the lung at the end of the experiment by histological method. The lung from the control mice showed an increase in the number of mononuclear cells infiltrates around bronchioli (mean number 1.5667.+-.0.89 respectively, versus 0.5333.+-.0.6756 in vaccination mice), and around blood vessels (mean number 0.8833.+-.0.8847 respectively, versus 0.3833.+-.0.6662 in vaccination mice) in comparison to the number of mononuclear cell infiltrates in the vaccination mice. Also control mice observed had significantly more patch cellular infiltrates than vaccination mice (mean number 1.3.+-.1.0939 respectively, versus 0.35.+-.0.6331 in vaccination mice).

Discussion

Diseases such as allergic asthma, rhinitis, and atopic dermatitis are all characterized by elevated levels of serum IgE. Total and specific IgE positively also showed a close relationship with clinical symptoms in atopic allergy. Analysis of antigen specificity of T-cell clones reactive with Der p 1 and Der p 2, with truncated recombinant proteins and synthetic peptides, has allowed several sites of T-cell recognition to be identified at different locations within Der p 1 and Der p 2. Several laboratories are now investigating the development of a new generation of immunotherapeutic strategies based on the modulation of T-cell function. Immunotherapy treatment has proven to be effective in treating some forms of allergy, but the antigen used was a poor immunogen and was needed at a higher concentration than the amount derived intracelluarly from processed antigens. Recently gene immunization with naked DNA was shown to suppress induction of IgE synthesis. These data suggest that immunization with a plasmid DNA (Pdna) containing the gene for the minor HDM allergen Der p 5 may induce Th1 immune responses to the encoded antigens. The Der p 5 allergen reacts with about 40% of allergic sera but the Der p 1 and 2 allergens react with about 80% of allergic sera..sup.19,20 It was examined whether plasmid DNA encoding T-cell epitopes of human Der p 1 and Der p 2 would also be able to induce abrogation of allergic responses in mice when they are given the naked DNA vaccine. We have analyzed the effects of gene vaccination with a plasmid encoding T-cell epitopes of Der p 1 (residues 45-67, and 94-143) and Der p 2 (residues 11-40, 61-104, and 111-129). Our results showed greater than 50% inhibition of total IgE and Der p-specific IgE at the end of the study (FIGS. 1A and 1B, see Original Patent). Thus, this result suggested that gene vaccination with plasmid DNA encoding the T-cell epitopes could also suppress induction of IgE synthesis.

To determine whether the suppressive effect of gene vaccination was due to the deletion of T-cells or the induction of unresponsiveness, we examined lymph node cell proliferation in response to different concentrations of Der p. We found that lymph node cells taken from BALB/c mice were able to respond by proliferation depending on the Der p concentration, with a similar pattern to that of the cells taken from the control mice. T-cell deletion has mainly been observed after administration of high doses of antigen or peptide. Furthermore, numerous experimental systems have shown that presentation of antigen by nonprofessional antigen presenting cells (APCs) that lack co-stimulatory capacity results in anergy rather than priming..sup.23 But professional APCs, Langerhans cells or macrophages, may act as APCS for intramuscular DNA vaccination. Also our results demonstrate strong T-cell responses to varying concentrations of Der p. Thus, gene vaccination did not induce T-cell deletion or anergy. The T helper 2 (Th2) cells mainly produce IL-4, IL-5 and IL-10 which induce antibody production in B cells, including above all, the formation of IgE which plays a central role in allergic responses. IFN-.gamma. is the Th1 cytokine responsible for the inhibition of IL-4-mediated IgE responses and promotes the formation of IgG2a. Protein immunization induces a Th2 response, as shown by IgG1 and IgE antibody formation and IL-4 and IL-5-secreting T-cells. In contrast, gene immunization with plasmid DNA induces a Th1 response with IgG2a antibody production and IFN-.gamma. secreting T-cell. To study the immune mechanisms involved in suppression of IgE synthesis after DNA vaccination, we measured the IFN-.gamma. and IL-4 serum levels in BALB/c mice after immunization of Der p extract. In the vaccinated mice there was an elevation in the Th1 cytokine IFN-.gamma. associated with suppression of IgE synthesis. In parallel, there was a reduction in the Th2 cytokine IL-4. Lee et al reported that the Th1 response dominated over the Th2 response and downregulated preexisting IgE antibody formation after genetic immunization. Our results showed that the genetic immunization with the plasmid DNA encoding only T-cell epitopes might also induce a Th2 to Th1 cytokine shift.

To study the effects of DNA vaccination on B cell immunity, we measured Der p specific serum antibodies. At 6 weeks after immunization, total serum levels of IgG2a Der p specific antibody increased and the Der p specific IgG1 response was reduced in the vaccination mice compared with control mice although the Der p specific IgG responses were similar between the two groups. IgG2a is dependent on interferon-.gamma. (IFN-.gamma.) as an IgM-to-IgG2a switch factor and is believed to be typical for a Th1 response. In contrast, IgG1 depends on IL-4 secreted by Th2 cells..sup.28 The Der p specific IgG isotype data further indicated that genetic vaccination with DNA encoding the T-cell epitopes induces a Th2 to Th1 cytokine shift, since vaccination group had increased IgG2a levels compared with the levels of the control group. Our results suggested that genetic immunization might suppress IgE production by inducing a Th2 to Th1 cytokine shift.

Allergic asthma is characterized as a chronic inflammatory disease of the bronchi and it is well established that a variety of cells including mast cells, eosionphils and lymphocytes play a role in this process. After inhalation challenge, the inflammatory cells migrate from the peripheral blood to the site of inflammation in the bronchial mucosa and bronchoalveolar fluid shows dominant Th2-type cytokines. Our histological evaluation revealed that a significant number of patch mononuclear cell infiltrates were observed around the bronchioles and blood vessels in the vaccination mice compared with the control mice. T lymphocytes have been suggested to play a key role in orchestrating the interaction of the participating cells since they are able to release an array of cytokines which can attract, prime and activate other cell types. A successful outcome of immunotherapy has been associated with the development of suppressor T-cells, which can downregulate the allergic response. It has been suggested that the change in the function of T-cells might cause a reduction in the number of inflammatory cells infiltrating lung tissue. This data indicated that gene immunization affects not only humoral immune responses but also cellular responses.

The vaccination with mixed naked DNA encoding only T-cell epitopes might induce abrogation of allergic response in mice as effectively as DNA encoding whole segment allergen. Thus gene therapy using DNA encoding T-cell epitopes could be an ideal way of combating allergic disease in the future.
 

Claim 1 of 8 Claims

1. A method comprising administering to a human, a composition comprising a pharmaceutically acceptable carrier and an isolated, naked DNA comprising a DNA fragment encoding a polypeptide consisting essentially of a portion of an immunogenic dust mite antigen to a human, wherein said DNA fragment is amplified by a primer pair selected from the group consisting of SEQ ID NO:45 and SEQ ID NO:46 for Der p 1, SEQ ID NO:47 and SEQ ID NO:48 for Der p 1, SEQ ID NO:49 and SEQ ID NO:50 for Der p 2, SEQ ID NO: 51 and SEQ ID NO:52 for Der p 2, SEQ ID NO: 53 and SEQ ID NO:54 for Der p 2, SEQ ID NO:1 and SEQ ID NO:2 for Der p 1, SEQ ID NO:3 and SEQ ID NO:4 for Der p 1, SEQ ID NO:5 and SEQ ID NO:6 for Der p 1 SEQ ID NO:7 and SEQ ID NO:8 for Der p 2, SEQ ID NO:9 and SEQ ID NO:10 for Der p 2, SEQ ID NO:21 and SEQ ID NO:22 for Der p 1 SEQ ID NO:23 and SEQ ID NO:24 for Der p 2, SEQ ID NO:25 and SEQ ID NO:26 for Der p 3, SEQ ID NO:27 and SEQ ID NO:28 for Der f 1, SEQ ID NO:29 and SEQ ID NO:30 for Der f 2, and SEQ ID NO:31 and SEQ ID NO:32 for Der f 3.

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