Nilsson Ola B, Neimert-Andersson Theresa, Bronge Mattias, Grundström Jeanette, Sarma Ranjana, Uchtenhagen Hannes, Kikhney Alexey, Sandalova Tatyana, Holmgren Erik, Svergun Dmitri, Achour Adnane, van Hage Marianne, Grönlund Hans
Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet, Stockholm, Sweden; Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Department of Clinical Neuroscience, Therapeutic Immune Design Unit, Center for molecular Medicine (CMM), Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2014 Oct 29;9(10):e111041. doi: 10.1371/journal.pone.0111041. eCollection 2014.
Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality.
To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog.
A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production.
The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses.
We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.
用于诊断和变应原特异性免疫治疗的犬皮屑提取物质量往往参差不齐且较差。
将四种成熟的犬变应原成分组装成一种重组折叠蛋白，以改善对犬过敏的诊断和疫苗接种。
构建了一种包含四种犬脂质运载蛋白变应原Can f 1、Can f 2、Can f 4和Can f 6的连接分子。通过小角X射线散射对四聚体蛋白进行结构表征，并通过分析型尺寸排阻色谱、圆二色性、ELISA检测血清中变应原特异性IgE以及变应原激活嗜碱性粒细胞的能力，将其与每种单一重组脂质运载蛋白变应原或四种变应原的等摩尔混合物进行比较。通过评估脾细胞增殖和抗体产生，在免疫小鼠中评估融合蛋白的免疫原性。
连接的四聚体构建体作为可溶性融合蛋白产生，四种单独变应原的特定折叠得以保留。这种多变应原分子在结合犬特异性IgE方面比每种单一重组变应原显著更有效（p<0.001），并且与四种变应原的等摩尔混合物相比，表位谱未受影响。嗜碱性粒细胞脱颗粒显示连接分子的生物学活性得以保留。用连接构建体免疫小鼠诱导出对所有包含变应原具有阻断能力的相当的变应原特异性IgG反应，并产生相当低的T细胞反应。
我们提供了首个证据，证明一种覆盖主要犬变应原的连接重组分子可用于犬过敏患者的诊断和过敏疫苗接种。
