A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

Abstract

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293 T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293 T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.

Keywords: DNA vaccine; Dermatophagoides allergen 1 group; asthma; chimeric gene.

Figure 1

Representation of the reporter and targeting vectors. PCMV (black arrow): Human cytomegalovirus immediate-early promoter; GFP (green box): enhanced green fluorescent protein; BGH pA (black box): The bovine growth hormone polyadenylation sequence. The diagrams are not drawn to scale.

Figure 2

Immunization schematic of the initiation of allergen-induced allergic airway inflammation in the mouse model and treatment with the DNA vaccine construct.

Figure 3

Detection of R8 gene expression in HEK 293T cells by fluoresent microscopy, 46 hours after treatment with the DNA-liposome complex (×200).

Figure 4

Western blot analysis of total proteins from pcDNA3.1-GFP-R8 transfected cells. In each lane, 80 µg of extracted protein was loaded. Lane 1: negative control (pcDNA3.1 vector transfected cells); lanes 2-4: total protein in cells transfected with plasmid pcDNA3.1-GFP, pcDNA3.1-GFP-ProDer f1, and pcDNA3.1-GFP-R8.

Figure 5

Pathological examinations of mouse lungs by H&E staining (×400). A: PBS group; B: asthma group; C: pcDNA3.1 group; D: pcDNA3.1-GFP group; E: pcDNA3.1-GFP-ProDer f1 group; F: pcDNA3.1-GFP-R8 group.

Figure 6

Detecting cytokines in the BALF of DNA vaccinated mice. Cytokines in the BALF from all animals were detected by quantitative ELISA. Data are shown as the mean ± SD of ten mice from each group. Note: a, compared with the PBS group, P < 0.001; b, compared with the asthma group, the pcDNA3.1 group, and the GFP group, P < 0.001; c, compared with the PBS group, P < 0.05; d, compared with the asthma group, the pcDNA3.1 group, and the GFP group, P < 0.05; e, compared with the ProDer f1 group, P < 0.05; f, compared with the ProDer f1 group, P < 0.001.

Figure 7

The levels of specific IgE, IgG₁, and IgG_2a in the sera of mice immunized with an R8-containing plasmid. Note: a, compared with PBS group, P < 0.001; b, compared with asthma group, pcDNA3.1 group, and GFP group, P < 0.001; c, compared with asthma group, pcDNA3.1 group, and GFP group, P < 0.05; d, compared with asthma group, P = 0.003.