Fel d 1-airway inflammation prevention and treatment by co-immunization vaccine via induction of CD4+CD25-Foxp3+ Treg cells

Abstract

Pet allergens are major causes for asthma and allergic rhinitis. Fel d 1 protein, a key pet allergen from domestic cat, can sensitize host and trigger asthma attack. In this study, we report that co-immunization with recombinant Fel d 1 protein (rFel d 1) plus plasmid DNA that contains Fe1 d 1 gene was effective in preventing and treating the natural Fel d 1 (nFel d 1) induced allergic airway inflammation in mice. A population of T regulatory cells (iTreg) exhibiting a CD4+CD25-Foxp3+ phenotype and expressing IL-10 and TGF-β was induced by this co-immunization strategy. Furthermore, after adoptive transfers of the iTreg cells, mice that were pre-sensitized and challenged with nFel d 1 exhibited less signs of allergic inflammation, AHR and a reduced allergic immune response. These data indicate that co-immunization with DNA and protein mixture vaccine may be an effective treatment for cat allergy.

Keywords: Fel d 1; T regulatory cell; airway inflammation; co-immunization; vaccine.

Figure 1. Schematic diagram of vaccine preparation. Vectors proVAX-rFel d 1 and pET28a-SMT3-rFel d 1 expressing rFel d 1 antigen were constructed as described in Materials and Methods. Vector proVAX-rFel d 1 was transformed in bacterial cells and plasmid DNA was purified and incorporated into a DNA vaccine. rFel d 1 was expressed in E.coli with IPTG induction, purified and used as a protein vaccine. Plasmid DNA was mixed 1:1 with the purified rFel d 1 to make co-immunization vaccine.

Figure 2. Lung inflammation and total serum IgE levels in mice inmmunized with different vaccines. (A) Schematic outline of the experiment. (B) Histological examination of lung tissues by H&E staining (n = 5). Magnification, × 200. (C) Lung inflammation was defined as the peribronchial inflammation score (n = 5). Values are expressed as mean ± SEM *** p < 0.001; significantly different from the allergic group. (D) Serum of individual mouse was collected after intratrachea with nFel d 1 (d42). Total serum IgE levels were measured using double-antibody sandwich ELISA technique (n = 6). * p < 0.05.

Figure 3. Prevention of AHR and acute systemic anaphylaxis by co-immunization strategy. (A) AHR was measured by the RC system in mice in response to the intra-jugular administration of acetylcholine chloride at various doses on the x-axis and expressed as Rrs on the y-axis. The allergic model was induced by nFel d 1 without vaccination (n = 6). (B) After vaccination and follow-up sensitization, 6 mice of each group were challenged i.v. with rFel d 1 and body temperature was measured at regular intervals. Changes in body temperature in degrees Celsius ± SD are shown on the left panel and mean area above the curves ± SD on the right panel (n = 6). Data shown are representative of three independent experiments with the same number of mice per group. A: * p < 0.05; B: ** p < 0.001.

Figure 4. Therapeutic relief of nFel d 1-induced allergic symptoms in mice by co-immunization. (A) Schematic outline of the experiment. (B) Histological examination of lung tissues (n = 5) by H&E staining (d43). Magnification, × 200. (C) Lung inflammation was defined as the peribronchial inflammation score (n = 5). Values are expressed as mean ± SEM * p < 0.05; significantly different from the control group. (D) Serum of individual mouse was collected after vaccination (d42). Total serum IgE levels were measured using double-antibody sandwich ELISA technique (n = 6) and shown as mean total serum IgE levels ± SD, ** p < 0.005.

Figure 5. Reduction of AHR and inhibition of anaphylactic reaction by DNA/Protein co-vaccination. (A) AHR was measured by the RC system in mice in response to the intra-jugular administration of acetylcholine chloride at various doses on the x-axis and expressed as Rrs on the y-axis. The nFel d 1-induced allergic model without vaccination were regarded as positive control (n = 6). (B) Eight days after the second vaccination, 6 mice in each group were challenged i.v. with rFel d 1 and body temperature was measured at regular intervals. Changes in body temperature in degrees Celsius ± SD (n = 6) are shown on the left panel and mean area above the curves ± SD (n = 6) on the right panel. Data shown are representative of three independent experiments with the same number of mice per group. (A): * p < 0.05; (B): ** p < 0.001.

Figure 6. Induction of CD4+CD25-Foxp3+ T regulatory cells in BALB/c mice by co-immunization with DNA and protein mixture (1:1) vaccine. (A) Schematic outline of the experiment. (B) Seven days after the second immunization, single splenocyte suspensions were prepared from mouse spleen and analyzed by flow cytometry. Percentages of CD4+CD25+Foxp3+ nTreg cells were analyzed by gating on the CD4+CD25+ T cells, and percentages of CD4+CD25-Foxp3+ iTreg cells were analyzed by gating on the CD4+CD25- T cells during FACS analysis. Naïve mice without vaccination were regarded as negative control. (C) Statistical analysis of percentages of nTreg cells (Left, n = 6) and iTreg cells (Right, n = 6). Compared with naïve group, mice treated with co-immunization strategy can induce CD4+CD25-Foxp3+ iTreg cells. Numbers in the outlined areas indicate percentage of cells. Data shown are representative of three independent experiments with the same number of mice per group. ** p < 0.005.

Figure 7. Increase in percentages of Foxp3+IL-10+ and Foxp3+TGF-β+ cells among CD4+CD25- cells in co-immunized mice. Seven days after the second immunization, single splenocyte suspensions were prepared and analyzed by flow cytometry. (A) Percentages of Foxp3+IL-10+ (upper) and Foxp3+TGF-β+ cells (lower) among CD4+CD25- cells. Percentages of double positive cells were shown. (B) Statistical analysis of percentages of Foxp3+IL-10+ cells and Foxp3+TGF-β+ cells in CD4+CD25- T cells (n = 3). Compared with other groups, percentages of double positive cells among CD4+CD25- T cells in the co-immunization group increased significantly (p < 0.05). Numbers in the outlined areas indicate percentage of cells. Data shown are representative of three independent experiments with the same number of mice per group. * p < 0.05.

Figure 8. Descrease in total serum IgE levels, reduction of AHR and inhibition of anaphylactic reaction by adoptively transfer CD4+CD25- T cells from co-immunized mice. CD4+CD25- T cells from co-immunized mice were isolated, purified and adoptively transferred intravenously to nFel d 1-induced allergic mice as described in Materials and Methods. Four different transfer groups received the following four doses of cells: 0.1 × 10⁶, 0.5 × 10⁶, 1.0 × 10⁶ and 5.0 × 10⁶. The nFel d 1-induced allergic airway inflammation mice without adoptive transfer served as a positive control. Naïve BALB/c mice without sensitization and transfer served as a negative control. (A) Schematic outline of the experiment. (B) 14 d after CD4+CD25- T cells transfer, serum of individual asthmatic mouse was collected. Mean total serum IgE levels ± SD are shown (n = 6). For 1.0 × 10⁶ recipient group vs. model group, p < 0.05; for 5.0 × 10⁶ recipient group vs. model group, p < 0.005. (C) AHR was measured by the RC system in mice in response to the intra-jugular administration of acetylcholine chloride at various doses on the x-axis and expressed as Rrs on the y-axis (n = 6). (D) 15 d after adoptive transfer, 6 mice in each group were challenged i.v. with rFel d 1 and body temperature was measured at regular intervals. Changes in body temperature in degrees Celsius ± SD (n = 6) are shown on the left panel and mean area above the curves ± SD (n = 6) on the right panel. Individual areas above the curves were determined and compared by one-way ANOVA using Bonferroni’s post test. Data shown are representative of three independent experiments with six mice per group. (B and C) * p < 0.05; ** p < 0.005; D: *** p < 0.0001; ns, no significant difference.

Figure 9. Effect of adoptive transfer of CD4+CD25- T cells from co-immunized mice on total serum IgE levels and AHR. CD4+CD25- T cells from different vaccine groups were isolated, purified and adoptively transferred intravenously to nFel d 1-induced allergic airway inflammation mice as described in Materials and Methods. Adoptively transferred CD4+CD25- T cells were 5.0 × 10⁶ cells per mouse. Four different transfer groups were set up [i.e., CD4+CD25- T cells were derived from naïve mice, BALB/c mice vaccinated with rFel d 1 alone, proVAX-rFel d 1 alone or proVAX-rFel d 1 and rFel d 1 mixture (1:1), respectively]. The nFel d 1-induced allergic mice without adoptive transfer served as positive control and the naïve BALB/c mice without sensitization and transfer served as blank control. (A) Schematic outline of the experiment. (B) Fourteen days after CD4+CD25- T cells transfer, serum of individual mouse was collected. Mean total serum IgE levels ± SD are shown (n = 6). (C) AHR was measured by the RC system in mice in response to the intra-jugular administration of acetylcholine chloride at various doses on the x-axis and expressed as Rrs on the y-axis (n = 6). Data shown are representative of two independent experiments with six mice per group. * p < 0.05; ** p < 0.005.