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The fold variant BM4 is beneficial in a therapeutic Bet v 1 mouse model.

Pichler U, Asam C, Weiss R, Isakovic A, Hauser M, Briza P, Ferreira F, Wallner M - Biomed Res Int (2013)

Bottom Line: Specific immunotherapy using recombinant allergens is clinically effective; still wild-type allergens can provoke treatment-induced side effects and often show poor immunogenicity in vivo.Further, birch pollen induced lung inflammation could be ameliorated significantly by BM4 treatment as demonstrated by a reduction of airway hyperresponsiveness and drastically decreased eosinophil counts in bronchoalveolar lavage fluids.The study outlines the high potential of BM4 as vaccine candidate for the treatment of Bet v 1-mediated birch pollen allergies.

View Article: PubMed Central - PubMed

Affiliation: Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, 5020 Salzburg, Austria.

ABSTRACT

Background: Specific immunotherapy using recombinant allergens is clinically effective; still wild-type allergens can provoke treatment-induced side effects and often show poor immunogenicity in vivo. Thus, we tested the low IgE-binding, highly immunogenic fold variant BM4 in a Bet v 1 mouse model.

Methods: Recombinant BM4 was used as active vaccine ingredient to treat mice sensitized to Bet v 1. As controls, mice were treated with either Bet v 1 or sham, and the humoral as well as cellular immune response was monitored. Moreover, lung function and lung inflammation were analysed.

Results: BM4 was more effective than wild-type Bet v 1 in inducing Bet v 1-specific blocking antibodies as well as IFN-γ and IL-10 producing T cells. Further, birch pollen induced lung inflammation could be ameliorated significantly by BM4 treatment as demonstrated by a reduction of airway hyperresponsiveness and drastically decreased eosinophil counts in bronchoalveolar lavage fluids.

Conclusion: The study outlines the high potential of BM4 as vaccine candidate for the treatment of Bet v 1-mediated birch pollen allergies.

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Related in: MedlinePlus

(a) Schematic representation of the therapeutic mouse model. (b) BALB/c mice were treated with either Bet v 1 (circles), BM4 (squares), or sham (triangles). Bet v 1-specific IgG levels were determined by ELISA, IgE by mediator release assays. Means ± SD are indicated, P-values were calculated with t-tests and paired-samples t-test, respectively (*P < 0.05, **P < 0.01).
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fig1: (a) Schematic representation of the therapeutic mouse model. (b) BALB/c mice were treated with either Bet v 1 (circles), BM4 (squares), or sham (triangles). Bet v 1-specific IgG levels were determined by ELISA, IgE by mediator release assays. Means ± SD are indicated, P-values were calculated with t-tests and paired-samples t-test, respectively (*P < 0.05, **P < 0.01).

Mentions: 8- to 10-week-old female BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and used for experiments 4 days after arrival (treatment schedule as shown in Figure 1(a)). All animal experiments were conducted according to the guidelines of the Austrian Ministry of Science (BMWF-66.012/0011-II/10b/2010). Six mice per group were sensitized subcutaneously (s.c.) with 5 μg Bet v 1 adsorbed to Alugel-S (Serva, Heidelberg, Germany) bilaterally in the lumbar region, followed by three intraperitoneal (i.p.) injections of 25 μg Bet v 1, or BM4 in PBS, or PBS alone. Aerosol challenges were performed with nebulized birch pollen extract (10 mg in 10 mL PBS) to induce airway hyperresponsiveness (AHR). ELISA and mediator release assays with murine sera were performed as described [2, 4]. In brief, for ELISA NUNC Maxisorp plates (Thermo Fisher Scientific, Waltham, USA) were coated with 2 μg/mL antigen solution over night at 4°C. Murine sera were applied in serial dilutions and incubated for 2 h at room temperature. Bound antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies (all from SouthernBiotech, Birmingham, USA) followed by chromogenic substrate development. Mediator release assays were performed using the cell line RBL-2H3 (ATCC number CRL-2256) passively sensitized with murine immune sera. After removal of the sera, antigen was added in serial dilutions and mediator release was determined by enzymatic cleavage of the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-β-glucosaminide (Sigma Aldrich, St. Louis, USA). β-hexosaminidase release is expressed as a percentage of the total enzyme content of Triton X-100-treated RBL-2H3 cells. All experiments were performed in duplicates.


The fold variant BM4 is beneficial in a therapeutic Bet v 1 mouse model.

Pichler U, Asam C, Weiss R, Isakovic A, Hauser M, Briza P, Ferreira F, Wallner M - Biomed Res Int (2013)

(a) Schematic representation of the therapeutic mouse model. (b) BALB/c mice were treated with either Bet v 1 (circles), BM4 (squares), or sham (triangles). Bet v 1-specific IgG levels were determined by ELISA, IgE by mediator release assays. Means ± SD are indicated, P-values were calculated with t-tests and paired-samples t-test, respectively (*P < 0.05, **P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3794650&req=5

fig1: (a) Schematic representation of the therapeutic mouse model. (b) BALB/c mice were treated with either Bet v 1 (circles), BM4 (squares), or sham (triangles). Bet v 1-specific IgG levels were determined by ELISA, IgE by mediator release assays. Means ± SD are indicated, P-values were calculated with t-tests and paired-samples t-test, respectively (*P < 0.05, **P < 0.01).
Mentions: 8- to 10-week-old female BALB/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and used for experiments 4 days after arrival (treatment schedule as shown in Figure 1(a)). All animal experiments were conducted according to the guidelines of the Austrian Ministry of Science (BMWF-66.012/0011-II/10b/2010). Six mice per group were sensitized subcutaneously (s.c.) with 5 μg Bet v 1 adsorbed to Alugel-S (Serva, Heidelberg, Germany) bilaterally in the lumbar region, followed by three intraperitoneal (i.p.) injections of 25 μg Bet v 1, or BM4 in PBS, or PBS alone. Aerosol challenges were performed with nebulized birch pollen extract (10 mg in 10 mL PBS) to induce airway hyperresponsiveness (AHR). ELISA and mediator release assays with murine sera were performed as described [2, 4]. In brief, for ELISA NUNC Maxisorp plates (Thermo Fisher Scientific, Waltham, USA) were coated with 2 μg/mL antigen solution over night at 4°C. Murine sera were applied in serial dilutions and incubated for 2 h at room temperature. Bound antibodies were detected with appropriate alkaline phosphatase-conjugated secondary antibodies (all from SouthernBiotech, Birmingham, USA) followed by chromogenic substrate development. Mediator release assays were performed using the cell line RBL-2H3 (ATCC number CRL-2256) passively sensitized with murine immune sera. After removal of the sera, antigen was added in serial dilutions and mediator release was determined by enzymatic cleavage of the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-β-glucosaminide (Sigma Aldrich, St. Louis, USA). β-hexosaminidase release is expressed as a percentage of the total enzyme content of Triton X-100-treated RBL-2H3 cells. All experiments were performed in duplicates.

Bottom Line: Specific immunotherapy using recombinant allergens is clinically effective; still wild-type allergens can provoke treatment-induced side effects and often show poor immunogenicity in vivo.Further, birch pollen induced lung inflammation could be ameliorated significantly by BM4 treatment as demonstrated by a reduction of airway hyperresponsiveness and drastically decreased eosinophil counts in bronchoalveolar lavage fluids.The study outlines the high potential of BM4 as vaccine candidate for the treatment of Bet v 1-mediated birch pollen allergies.

View Article: PubMed Central - PubMed

Affiliation: Christian Doppler Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, 5020 Salzburg, Austria.

ABSTRACT

Background: Specific immunotherapy using recombinant allergens is clinically effective; still wild-type allergens can provoke treatment-induced side effects and often show poor immunogenicity in vivo. Thus, we tested the low IgE-binding, highly immunogenic fold variant BM4 in a Bet v 1 mouse model.

Methods: Recombinant BM4 was used as active vaccine ingredient to treat mice sensitized to Bet v 1. As controls, mice were treated with either Bet v 1 or sham, and the humoral as well as cellular immune response was monitored. Moreover, lung function and lung inflammation were analysed.

Results: BM4 was more effective than wild-type Bet v 1 in inducing Bet v 1-specific blocking antibodies as well as IFN-γ and IL-10 producing T cells. Further, birch pollen induced lung inflammation could be ameliorated significantly by BM4 treatment as demonstrated by a reduction of airway hyperresponsiveness and drastically decreased eosinophil counts in bronchoalveolar lavage fluids.

Conclusion: The study outlines the high potential of BM4 as vaccine candidate for the treatment of Bet v 1-mediated birch pollen allergies.

Show MeSH
Related in: MedlinePlus