Protective effect of the DNA vaccine encoding the major house dust mite allergens on allergic inflammation in the murine model of house dust mite allergy

Background Vaccination with naked DNA encoding antigen induces cellular and humoral immunity characterized by the activation of specific Th1 cells. Objective To evaluate the effects of vaccination with mixed naked DNA plasmids encoding Der p 1, Der p 2, Der p 3, Der f 1, Der f 2, and Der f 3, the major house dust mite allergens on the allergic inflammation to the whole house dust mites (HDM) crude extract. Methods Three hundred micrograms of these gene mixtures were injected into muscle of BALB/c mice. Control mice were injected with the pcDNA 3.1 blank vector. After 3 weeks, the mice were actively sensitized and inhaled with the whole house dust mite extract intranasally. Results The vaccinated mice showed a significantly decreased synthesis of total and HDM-specific IgE compared with controls. Analysis of the cytokine profile of lymphocytes after challenge with HDM crude extract revealed that mRNA expression of interferon-γ was higher in the vaccinated mice than in the controls. Reduced infiltration of inflammatory cells and the prominent infiltration of CD8+ T cells were observed in histology of lung tissue from the vaccinated mice. Conclusion Vaccination with DNA encoding the major house dust mite allergens provides a promising approach for treating allergic responses to whole house dust mite allergens.


Background
It has been reported that IgE-mediated inflammation to mites is associated with diseases such as asthma, allergic rhinitis, and atopic dermatitis and that the relatively minor determinants of house dust mite (HDM) allergens show IgE binding with 40% or more of allergic sera [1][2][3][4].
The most frequently implicated allergens are derived from Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) [5]. Targeting specific T lymphocytes that induce or regulate the allergic inflammation is one of the therapeutic goals in allergic disorders. Specific immunotherapy with crude extracts has been used mainly in treating HDM-induced allergy. However, it has limited efficacy. Recently, vaccinations with naked DNA encoding antigen were reported to induce long-lasting cellular and humoral immune tolerance [6,7]. Injection of plasmid DNA encoding T cell epitopes could suppress allergic reaction [8,9]. However, the potential barrier to T cell receptor-based immunotherapy for allergy is the apparent complexity of the allergen-specific T cell response in terms of epitope usage in individuals [10]. A recent literature search showed that injection of plasmid DNA encoding Der p 5 in rat not only inhibits Der p 5-specific IgE antibody production but also an allergic response such as histamine release and airway hyperresponsiveness to Der p 5 [11]. However, Der p 5 is not one of the major HDM allergens; thus, it has limited clinical application [3]. In this study, we investigated immune responses by gene vaccination with plasmid DNA encoding major HDM allergens (Der p 1,2, and 3, and Der f 1,2, and 3) to challenges with whole HDM crude extract in sensitized mice.

Animals
Twenty female BALB/c mice 6-8 weeks old were purchased from Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility of the University of Tennessee Health Science Center. This study was performed in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals and 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.

DNA preparation and vaccination
Each plasmid construct was prepared using Maxi prep (Quiagen, Chatsworth, CA). Mice were vaccinated by injection with 300 µg of pcDNA3.1 blank vector in 100 µl of phospate-buffered saline (PBS) (the control group) or the same amount of the mixed naked DNA encoding the major HDM allergens (the vaccination group) three times at weekly intervals into muscle (week 0, 1, and 2).

Immunization and inhalation of allergen
Mice were sensitized with HDM crude extract previously described [12]. HDM crude extract was emulsified with an equal volume of complete Freund adjuvant (CFA) for immunization. Three weeks after the last vaccination, mice were sensitized subcutaneously at the base of the tail with 100 µg of HDM extract in CFA. The mice were also given an intraperitoneal dose of 300 ng of purified pertussis toxin at 24 and 72 h after first immunization. Seven days later, the mice were boosted again with the same amount of antigen in incomplete Freund adjuvant. Under inhaled anesthesia with methoxyflurane, mice were challenged with 10 µg of HDM crude extract through one nostril six times at weekly intervals after immunization.

Determining total IgE, HDM-specific IgE, and HDMspecific IgG
The blood from the six mice in two groups was collected six times at week 0 (first vaccination), 3, 5 (first immunization), 7, 9, and 11. The HDM-specific IgG was determined by ELISA. One hundred microliter of HDM (5 µg/ ml in 0.

Immunohistochemical staining for CD4+ and CD8+ T cells in lung tissue
The lung tissues from the vaccination and control groups were removed immediately after the final intranasal inhalation.

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 PBS and then with 4% paraformaldehyde 0.1 M PBS buffers. Whole lungs were taken out and stored in 4% paraformaldehyde for 24 h at 4°C. After fixation, these tissues were dehydrated and embedded in paraffin. Frozen sections cut at 3 µm in thickness were stained by hematoxylin and eosin.
After coding, the sections were evaluated by two observers using light microscopy. The amount of inflammation per section was scored using the method described by Hessel et al. [13]. Lungs that showed no focal inflammation were scored as grade 0. Those that showed one or two centrally located microscopic foci of inflammatory infiltrate were graded as 1. In grade 2, a dense inflammatory infiltrate was seen in a perivascular and peribronchial distribution originating in the center of the lung. In grade 3, the perivascular and peribronchial infiltrates extended to the periphery of the lung.

Measuring cytokine mRNA expression
Measuring the expression level was done as previousy described [9]. Briefly, four mice from each group were sacrificed 10 days postboost. The lymph nodes were removed from the mice and minced to create single cell suspensions. Cells were cultured in RPMI for 18 h with no antigen as a negative control, recombinant Der p 1 (100 µg/ ml), or HDM crude extract (100 µg/ml). Cells were washed with PBS buffer and mRNAs prepared (Biotech, Houston, TX). 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, Branchber, NJ), first-strand cDNA was generated from 1 µg of total RNA and subjected to RT-PCR analysis. We used the primers specific for β-actin  Figure 1 Effect of vaccination on immunoglobulin production. The total IgE antibody levels (1a), HDM-specific IgE antibody levels (1b), and HDM-specific IgG antibody levels (1c) in sera of each mouse were detected by ELISA every 2 weeks after immunization with HDM. The data are expressed as means ± SD (n = 6 per group). *P < .05 compared with the control group.

Statistical analysis
Data in immunoglobulin response were analyzed by Student's paired t test for comparisons between control and vaccination groups. Histological grades were analyzed by a nonparametric Mann-Whitney U test. Data were expressed as mean ± SD. A p-value of < 0.05 was considered significant.

Downregulation of allergen specific IgE production by DNA vaccination
DNA vaccination with the major HDM allergen gene, Der p 1, 2, and 3, and Der f 1, 2, and 3 showed about 50% reduction of HDM-specific IgE and more than 70% reduction of total IgE compared with the control group at 6 weeks after immunization ( Fig. 1a and 1b). However, pro-duction of HDM-specific IgG antibody showed no difference (Fig. 1c). Thus, in vivo total and allergen-specific IgE synthesis might be efficiently inhibited by DNA vaccination.

Histological and immunohistochemical study
To investigate whether the DNA vaccination affects inflammation of lung, we stained lung tissue by histological and immunohistochemical methods. The lungs from the control group showed much more infiltration of inflammatory cells in the submucosa of airways than did those lungs from the vaccination group. The inflammation grades were scored as 1.64 ± 0.52 (mean ± SD) in the control group and 0.68 ± 0.48 in the vaccination group (Fig. 2a, 2b, and 2c). Also, eosinophils were detected in the lungs of the control mice (Fig. 2d). In the immunohis-Effect of genetic vaccination on lung histopathology in an animal model of allergy Figure 2 Effect of genetic vaccination on lung histopathology in an animal model of allergy. A and B, Light microscopic examinations of lung tissue from control group mouse (×100 and ×200). C, From vaccination group mouse (×200). D, Inflammatory cells including eosinophils (indicated with arrow) were observed in the peribronchial area in lung tissue from control group (×600). All tissue samples were stained with hematoxylin and eosin.
tochemical stain for CD4 and CD8 molecules, the more CD8+ cells were infiltrated in the submucosa and mucosa of airway from the vaccination group compared with the control group (Fig. 3). But no difference in CD4+ cells was shown between the two groups. We considered whether the DNA vaccination might have an effect on the cellular response and the CD8+ T cells, which might protect against subsequent allergen challenges.

Cytokines expressed by antigen stimulation
Lymphocytes were harvested from lymph nodes of the two groups of mice and stimulated with recombinant Der p 1 or HDM crude extract to determine the Th1 or Th2 cytokines involved in the DNA vaccination. Significantly elevated expression of IFN-γ mRNA was detected in the vaccination group compared with that in the control group. However, mRNA expression of IL-2, 4, 5, and 10 showed no difference from the control group (Fig. 4).

Discussion
Diseases such as allergic asthma, rhinitis, and atopic dermatitis are all characterized by elevated levels of serum IgE. Total and specific IgE levels also show a close relationship with clinical symptoms in atopic allergy [14]. A variety of approaches targeting the suppression of IgE have been proposed such as synthetic peptides and T cell vaccine. However, synthetic peptides have substantial limitations because of poor immunogenecity [15,16]. Recently, in an animal model of allergic disorders, DNA vaccine encoding one of the major birch pollen allergens has been shown to be allergen-specifically protective and therapeutic [17]. DNA vaccination with plasmid encoding Der p 5, Figure 3 Immunohistochemical examination for CD8+ T cells in lung tissue. In the lung tissue from the vaccination group, more CD8+ T cells were infiltrated along the airway than in control group. A and B, Lung tissue from control group mouse (×100 and ×200). C and D, Vaccination group (×100 and ×200). Immunohistochemical staining with rat anti-mouse CD8 monoclonal antibody.

Immunohistochemical examination for CD8+ T cells in lung tissue
one of the minor HDM allergens, was reported to prevent induction of specific IgE synthesis [11]. Vaccination with pDNA encoding Der p 5 was shown to induce Th1 immune response to the encoded antigens. However, these results have some limitations on the clinical application for treating allergic disorders. Each allergen that causes allergic disorders in humans contains various kinds of protein that have their own epitopes and are complex. The Der p 5 allergen reacts with about only 40% of allergic sera to HDM, and the Der p 1 and 2 allergens react with about 80% of allergic sera [3,5]. To evaluate the effect of the gene vaccination with DNA fragments encoding major allergen on the allergic response to whole HDM extract, we used plasmid with cDNAs encoding the major six HDM allergens (Der p 1, 2, and 3, and Der f 1, 2, and 3) for vaccination. We showed about 50% reduction of HDM-specific IgE and more than 70% inhibition of total IgE at 6 weeks after immunization compared with control group. DNA vaccine with plasmid encoding the major HDM allergens might inhibit IgE synthesis more efficiently than encoding one of the HDM allergens.
In animal models of allergic disease, it has been established that Th2 responses are mediated by T helper cells that secret cytokines such as IL-4 and IL-5, which induce antibody production in B cells, and IgE plays a central role in allergic responses [18]. IFN-γ is the Th1 cytokine responsible for inhibiting IL-4-mediated IgE responses and promoting the formation of IgG2a [19]. Plasmid vector containing DNA that encodes allergens has been reported to decrease Th2-mediated responses, enhance Th1 responses, and suppress the allergic response [11,20,21]. In this investigation, mRNA expression of IFN-γ in lymphocytes from the vaccination group increased significantly relative to that from the control group, and less production of total and specific IgE in the vaccinated group was detected than in the control group, suggesting that the gene vaccination might successfully redirect the immune response from Th2 into Th1 to the encoded antigen or allergen.
Allergic asthma is characterized as a chronic inflammatory disease of the bronchi. It is well established that a variety of cells including mast cells, eosinophils, and lymphocytes play a role in this process [22,23]. After inhalation challenges, the inflammatory cells migrate from the peripheral blood to the site of inflammation in the bronchial mucosa, and Th2 type cytokines are dominantly detected in bronchoalveolar lavage fluid [24,25]. However, our investigation showed that DNA vaccination successfully reduced the recruitment of inflammatory cells in lung tissues. The effect of DNA vaccination in allergic inflammation might be elicited through not only humoral immune responses but also cellular responses.
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 that can attract, prime, and activate other cell types [25]. A successful outcome of immunotherapy is known to be associated with the development of regulatory T cells, which can downregulate the allergic response [26][27][28][29][30]. It is also known that functionally distinct subsets of CD8+ T cells may play an important regulatory role in IgE production [30][31][32]. However, there are some different explanations regarding the mechanisms of DNA vaccine. Manickan et al. [33] demonstrated the mechanism of genetic immunization against herpes simplex virus principally by CD4+ T cells, not by CD8+ T cells. Lee et al. [22] reported that both CD4+ and CD8+ subsets of T cells from pDNA vaccinated mice can suppress IgE antibody production by affecting the primary response or by propagating the Th1 memory Cytokine expression in lymphocytes Figure 4 Cytokine expression in lymphocytes. Lymphocytes from the control group and vaccination group were cultured in the presence of no antigen (N), recombinant Der p 1 (P1), and HDM crude extract (HDM) for 18 h. mRNAs of each indicated cytokine (interferon-gamma, IL-2, IL-4, IL5, IL-10) were measured by RT-PCR and that of beta-actin was measured for control.
response in a passive cell transfer system. Draghi et al. [34] investigated whether DNA vaccination leads to the generation of a distinct population of noncytotoxic/regulatory CD8+ T cells. In the authors' immunohistochemical investigation, more CD8+ T cells were more infiltrated in the lung tissue of the vaccination group than that of the control group.
Peptides derived from extracellular molecules are presented to CD4+ T cells by MHC (histocompatibility complex) class II molecules normally generated by antigenpresenting cells, whereas peptides derived from intracellular proteins are generally presented to CD8+ T cells by MHC class I molecules, which are expressed on virtually all somatic cells [35]. We injected mixed naked DNA into the muscle of the murine model of allergic disorder. Some of the injected DNA might be postulated to stay in the nuclei of cells or be integrated in the host DNA and elicit the endogenous production of an allergenic protein.
MHC class I molecule, might induce CD8+ T cells that protectively function in immune response against a subsequent allergic challenge in sensitized host cells. The CD8+ T cells might be capable of conferring protection from allergic inflammation. DNA vaccination, which contains plasmid and DNA encoding specific allergen, might provide a more efficient therapeutic method for intervening allergic responses than conventional specific immunotherapy with allergen extracts.