Immunogenicity of a novel anti-allergic vaccine based on house dust mite purified allergens and a combination adjuvant in a murine prophylactic model

Abstract

The outer-membrane-derived proteoliposome (PL) of Neisseria meningitidis has been reported as a potent vaccine adjuvant, inducing a Th1-skewed response. This work aimed to assess the immunogenicity of a novel anti-allergic vaccine candidate based on allergens from Dermatophagoides siboney house dust mite and a combination adjuvant containing PL and Alum. In a preventative experimental setting, BALB/c mice were administered with three doses containing 2 µg of Der s1 and 0.4 µg Der s2 allergen, PL and Alum, at 7 days intervals, by subcutaneous route. Furthermore, mice were subjected to an allergen aerosol challenge for 6 consecutive days. Serum IgE, IgG1, and IgG2a allergen-specific antibodies were assessed by ELISA. Cytokine levels in supernatants of D. siboney stimulated lymphocyte cultures and in bronchoalveolar lavage (BAL) were measured by ELISA. Lung tissues were subjected to histological examination. The vaccine prevented the development of both, systemic (IgE) and local allergic responses (featuring lower IL-4, and IL-5 levels in BAL) upon allergen exposure by the inhalant route. Histological examination showed also a diminished allergic inflammatory response in the lungs. After the allergen challenge, cytokine levels in stimulated lymphocyte cultures showed lower values of IL-13 and augmented IFN-γ and IL-10. The vaccine induced a mixed IgG2a/IgG1 antibody response; although only IgG2a was PL-dependent. Both, IgG1/IgE and IgG2a/IgE ratios, showed significantly greater values in vaccinated mice. The findings support a preventative anti-allergic effect associated with the induction of a Th1-like IFN-γ/IL-10 response. IgG1/IgE and IgG2a/IgE ratios could be useful biomarkers for translation into clinical trials.

Keywords: Dermatophagoides siboney; TLR ligands; adjuvanted vaccine; allergy immunotherapy; combination adjuvant.

Figure 1

Allergen-specific antibody response, (A) IgG1, (B) IgG2a, (C) IgE, to vaccine and placebos (alum in PBS or PBS alone), as well as, in the Th2 control group injected with an allergen formulation adsorbed into alum lacking the PL adjuvant, at 0, 14, and 21 days after the beginning of the immunization. Values are shown as mean ± SD from n = 10/group. (***p < 0.001; 2-way ANOVA, Bonferroni test).

Figure 2

Allergen-specific antibody response upon allergen challenge: before (at 21 days) and after (at 28 days) the challenge. (A) IgG1, (B) IgG2a, (C) IgE. Values are shown as mean ± SD from n = 10/group. (p-values from 2-way ANOVA, Bonferroni test). The vaccinated mice showed an increase of IgG1 as well as a moderate increase of IgE, the Th2 control showed an exacerbated IgE response. PBS was used as the placebo. (*p < 0.05; ***p < 0.001; 2-way ANOVA, Bonferroni test).

Figure 3

Allergen-specific IgG/IgE ratios and IgG1/IgG2a ratio upon allergen challenge: before (at 21 days) and after (at 28 days) the challenge. Values are shown as mean ± SD from n = 10/group. (p-values from 2-way ANOVA, Bonferroni test). (A) Allergen-specific IgG1/IgE ratio, (B) Allergen-specific IgG2a/IgE ratio, (C) Allergen-specific IgG1/IgG2a ratio. The vaccinated mice showed a significant difference as compared with the Th2 control group prior to the challenge and kept this significant difference after the challenge. (***p < 0.001; 2-way ANOVA, Bonferroni test).

Figure 4

Eosinophil counting in peripheral blood, in mice subjected to the allergenic challenge. Vaccinated mice show a diminished eosinophil response. Letters a, b, and c indicate significant differences. PBS was used as the placebo. Values are shown as mean ± SD from n = 10/group (p < 0.05, ANOVA, Tukey test).

Figure 5

Cytokine levels after allergen challenge in supernatants of lymphocyte cultures stimulated with the allergen (A) and in BAL (B). In lymphocyte culture, vaccinated mice showed augmented values of IFN-γ and IL-10 and diminished IL-13 levels as compared with the Th2 control group. In BAL, vaccinated mice showed also increased IFN-γ levels and decreased Th2 inflammatory cytokines (IL-4 and IL-5). PBS was used as the placebo. Values are shown as mean ± SD from n = 5/group. (*p < 0.05; ***p < 0.001; 2-way ANOVA, Bonferroni test).

Figure 6

Representative lung histology sections after the allergen challenge. The lungs were stained by hematoxylin-eosin (200×). (A) Th2 control group depicting allergic response with peribronchial inflammation characterized by bronchial hyperreactivity features (defined by bronchoconstriction and stenosis with hyperplasia of the epithelial cells) as well as vascular and septal infiltrative changes with a predominance of lymphocytes and eosinophils. (Indicated by arrows) (B) Vaccinated mice depicting a discrete allergic inflammatory response characterized by a slight thickening of the bronchial wall. (C) Non-vaccinated non-challenged mice as the negative control.

Figure 7

Influence of previous immunization with N. meningitidis PL into allergen-specific (left) and N. meningitidis-specific (right) IgG1 and IgG2a antibody response. Double vaccinated mice (i.e., mice administered first with PL and later with the allergen PL-adjuvanted vaccine) showed no change in anti-N. meningitidis antibodies as compared with single vaccinated mice (PL alone or allergen + PL alone), though a significant boost of IgG1 allergen-specific antibodies (*p < 0.05, ANOVA). Values are shown as mean ± SD from n = 10/group.