The influence of antigen targeting to sub-cellular compartments on the anti-allergic potential of a DNA vaccine

Abstract

Background: Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments.

Methods: DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined.

Results: Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response.

Conclusion: This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special emphasis on prophylactic anti-allergy DNA vaccination. Targeting the antigen to proteasomal or lysosomal degradation reduces the availability of native allergen, thereby rendering the vaccine hypoallergenic without compromising efficacy, an important feature for a therapeutic setting.

Keywords: 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II; AA; AHR; APC; BAL; BALF; DNA vaccine; ER-targeting; Immunotherapy; LIMP-II; LIMPII; SIT; T helper; T regulatory; Th; Treg; Type I allergy; Ubi; Ubiquitination; airway hyperreactivity; amino acid; antigen presenting cell; bronchoalveolar lavage; bronchoalveolar lavage fluid; human tissue plasminogen activator leader peptide; specific immunotherapy; tPA; ubiquitin.

Fig. 1

DNA vaccine-targeting strategies. After entering the nucleus (1), the plasmid DNA is transcribed and differentially processed, depending on the respective modification. (a) The 5 ‘attached tPA signal sequence leads to shuttling of the vaccine-derived Bet to the exterior of transfected cells via the Golgi apparatus, while (b) the unmodified genetic information is translated into the cytosol, leading to vaccine-derived endogenous peptide presentation on MHC-I molecules. (c) Ubiquitin attachment feeds the translated protein into the polyubiquitination pathway thereby specifically targeting peptides to MHC-I. (d) In contrast, LIMPII peptide attachment promotes the antigenic transport to lysosomes that facilitate peptide presentation on MHC-II. Along with direct transfection of both, resident immunocompetent as well as somatic cells, (2) the engulfment of secreted vaccine-derived antigens, that have been shed from transfected cells, enforce peptide processing within the endocytic pathway, or, (3) MHC-I cross-presentation of cell-associated exogenous antigens, e.g. by engulfment of transfected and apoptotic cells, are potential modes of neoantigen presentation to the immune system.

Fig. 2

Bet-specific humoral and cellular immune responses upon i.d. genetic vaccination. (A) Schematic overview of the experimental schedule. Mice were i.d. immunized (triangle) in weekly intervals and blood samples (drop) were taken at day 49 after initial immunization. Bet-specific IgG1 (B) and IgG2a (C) antibody levels 5 weeks after the final vaccination were determined by luminescence-based ELISA. Depicted are results at a final sera dilution of 1:1000. (D) Proliferation of in vitro Bet re-stimulated splenocytes was assessed via ³H thymidin incorporation and the number of IFN-γ spot forming units (SFU) was determined by ELISPOT assay (E). Data are shown as means ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared to naïve animals or as indicated.

Fig. 3

Humoral immune profile after vaccination and sensitization. (A) Schematic overview of the experimental schedule. Mice were i.d. immunized (black triangle) three times in weekly intervals and sensitized (gray triangle) for three times, before blood samples (drop) were taken at day 56 after initial immunization. After three consecutive allergen inhalation challenges (gray trapezium), lung resistance/compliance was measured and mice were sacrificed (cross). Bet-specific IgG1 (B), IgG2a (C) and IgE (D) antibody levels 1 week after sensitization (day 56) were determined via luminescence-based ELISA (IgG1, IgG2a) or RBL assay (IgE). Control animals received sham immunizations (empty pCI vector; mock) or no pre-vaccination (control) prior to Bet protein sensitization. Sera were diluted 1:1000 for ELISA (B and C) and 1:50 for RBL (D). For BAT, whole blood was ex vivo stimulated with Bet protein. Data are displayed as fold induction of up-regulated CD200R of antigen-stimulated vs. un-stimulated basophils. Data are shown as means ± SEM (n = 6). *P < 0.05; **P < 0.01; ***P < 0.001 compared to control group or as indicated.

Fig. 4

Cellular proliferation and cytokine responses of Bet re-stimulated splenocytes from pre-vaccinated animals, sham immunized (mock), or non-immunized (control) animals after sensitization. (A) Numbers of IL-4 and IFN-γ secreting splenocytes (ELISPOT) as well as an extensive panel of other cytokines (B–G) released from Bet re-stimulated splenocytes (FlowCytomix) were measured. (H) CFSE-based analysis of proliferating CD4+ T cells, given as fraction of proliferating to non-proliferating cells as well as (I) the percentage of CD25+ Foxp3+ of proliferating CD4+ T cells are displayed. Data are shown as means ± SEM (n = 6 or 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared to control group or as indicated.

Fig. 5

Airway hyperresponsiveness and BAL analysis of Bet pre-vaccinated, sham-immunized (mock), or non-immunized (control) animals following sensitization and airway challenge. AHR was assessed after Bet inhalation by measurement of lung resistance (A) and dynamic compliance (B). Cellular composition of BAL (C–G) was analyzed via flow cytometric analysis and BALF cytokine levels (H–J) were assessed by FlowCytomix. Both assays are presented as individual data points and/or means ± SEM (n = 6 or 3). AUC, area under curve; *P < 0.05; **P < 0.01; ***P < 0.001 compared to control group or as indicated.