Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model

Background

Allergen‐specific immunotherapy can induce long‐term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as β‐glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment.

Aims

Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)‐driven mouse model of allergic asthma.

Materials and methods

HDM‐sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen‐induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue.

Results

ESR measurement shows suppression of early allergic response in HDM‐SCIT– and DerP1/2‐SCIT–treated mice. Both HDM‐SCIT and DerP1/2‐SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2‐SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2‐SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL‐33, CCL17 and eotaxin levels in lung tissue.

Discussion

Taken together, these data show that purified DerP1/2‐SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM‐induced activation of lung structural cells including airway epithelium.

Conclusions

We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.     

The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response

The house dust mite Dermatophagoides pteronyssinus allergen Der p 1 is the most immunodominant allergen involved in the expression of dust mite-specific immunoglobulin (Ig)E-mediated hypersensitivity. The reason for this potent IgE-eliciting property of Der p 1 remains unknown, but there is mounting in vitro evidence linking the allergenicity of Der p 1 to its cysteine protease activity. Here we demonstrate for the first time that immunization of mice with proteolytically active Der p 1 results in a significant enhancement in total IgE and Der p 1-specific IgE synthesis compared with animals immunized with Der p 1 that was irreversibly blocked with the cysteine protease inhibitor E-64. We conclude that the proteolytic activity of Der p 1 is a major contributor to its allergenicity.     

空调空气滤网灰尘中尘螨变应原Derfl和Derpl基因的检测

目的应用PCR技术检测空调空气滤网灰尘中尘螨变应原Derfl和Derpl的基因。方法从深圳和北京地区分别收集20台、10台空调过滤网上的灰尘样品，分别提取其生物总基因组DNA作PCR模板。分别设计Derfl和Derpl各两套内外扩增引物分两步进行PCR，通过扩增出目的片段来检测空调空气滤网灰尘中尘螨变应原Derfl和Derpl基因片段，并以深层泥土DNA提取物为阴性对照和以培养的粉尘螨、屋尘螨基因组DNA提取物为阳性对照。PCR产物经电泳后切胶回收日的片段并克隆到T载体上，进行DNA序列分析并与GenBank进行同源性比较分析。结果深圳地区20台空调空气滤网上收集的灰尘中均含有粉尘螨和屋尘螨主要变应原Derfl和Derpl基因片段；北京地区10台空调空气滤网上收集的灰尘样品中，只有4份均含有Derfl和Derpl；它们扩增部分的序列与GenBank数据库里相应序列同源性为100％。从阴性对照提取物中未扩增得到目的变应原Derfl和Derpl基因片段，从阳性对照的DNA提取物中分别扩增得到目的交应原Derfl和Derpl基因片段。结论本研究首次应用分子生物学技术证实了空调空气滤网上的灰尘中存在尘螨主要变应原Derfl和Derpl基因，并提示深圳地区空调过滤网灰尘中尘螨可能比北京地区的多，从而为今后防治空调中尘螨引起的变态反应性疾病提供了依据。     

哮喘患者居室内尘螨孳生种类、密度及其与抗原浓度的相关性研究

目的调查海口市哮喘患者居室孳生尘螨种类、密度及其与抗原浓度的相关性。方法采集哮喘患者居室尘样,在光镜下分类、计数,并用ELISA法测定Der P1浓度。结果制片616张,鉴定出16种螨,其中热带无爪螨占71.75%,屋尘螨占9.25%。哮喘患者居室尘样螨孳生密度和抗原浓度均高于正常对照组,差异均有统计学意义(P<0.01),二者间相关性显著(r=0.716,P<0.01)。结论热带无爪螨为本地区优势螨种,居室尘螨抗原浓度的检测似可替代活螨计数法评价室内尘螨孳生情况。     

屋尘螨变应原Der p 1基因的克隆和原核表达

目的克隆和分析屋尘螨主要变应原Der p 1,并实现原核表达。方法分离屋尘螨总RNA,根据Gen Bank已公布的Der p 1核酸序列设计引物,用RT-PCR扩增Der p 1编码基因,克隆至pMD19-T载体、亚克隆至表达载体pET-28a( ),将表达质粒转化至E.coliBL21(DE3)并用IPTG诱导表达,并对表达产物进行免疫印迹鉴定。结果成功构建了表达质粒pET-28a( )-Der f 1,Western blotting显示原核表达获得成功。序列分析表明所获得的Der p 1编码基因与参考序列同源性达99.9%,推测其编码氨基酸222个。屋尘螨和粉尘螨1类变应原氨基酸序列相似率为60%,而粉尘螨与梅氏嗜霉螨1类变应原相似率为85%,分子进化分析亦提示粉尘螨和梅氏嗜霉螨亲缘关系较近。推测所获rDer p 1二级结构中,α螺旋占33.78%,延伸链占21.62%,随机线圈占44.59%。结论尘螨变应原Der p 1原核表达获得成功,为进一步生产重组变应原奠定了基础。序列分析表明粉尘螨和梅氏嗜霉螨的亲缘关系可能更近,而与屋尘螨关系稍远,此与现行的形态学分类系统并不符合。     

Molecular mimicry as the key to the dominance of the house dust mite allergen Der p 2

Evaluation of: Trompette A, Divanovic S, Visintin A et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 457, 585–588 (2009).

The majority of complex sources of allergen contain a small number of dominant allergens that bind at least half of the IgE antibodies of allergic subjects. For the house dust mite Dermatophagoides pteronyssinus, these allergens are Der p 1 and Der p 2. There is evidence that the cysteine-protease activity of Der p 1 imparts adjuvant activity to the allergen. It has now been shown that Der p 2 mimics the activity of its fellow ML-domain protein, MD-2, by presenting lipopolysaccharide to Toll-like receptor-4 for the activation of inflammatory genes. In accord with this, Der p 2 presented with lipopolysaccharide also induced enhanced type 1 allergic sensitization of mice, even when they were deficient in MD-2. The mimicry of MD-2 can thus also self adjuvant Der p 2 to enhance it allergenicity. This not only describes an intriguing mechanism for enhancing allergenicity but also, since both of the dominant mite allergens have intrinsic adjuvant activity, exemplifies an important principle for driving allergic sensitization.     

Differences in titres of IgE, IgG4 and other IgG subclass anti- Der p 2 antibodies in allergic and non-allergic patients measured with recombinant allergen

Background The mite allergens are recognized as major causes of allergic disease such as bronchial asthma, allergic rhinitis and atopic dermatitis. The functions of allergen-specific IgG subclass antibodies are not defined.
Objective In order to clarify the relationship between IgE and IgG subclasses, we examined scrum levels of the Dermatophagoides pteronyssisus group 2 (Der p 2)-specific antibodies of IgH. IgG total and IgG subclasses in children with mite allergy.
Methods We prepared a recombinant Der p 2 fusion protein and examined serum levels of Der p 2 antigen-specific antibodies by enzyme-linked immunosorbent assay (FLISA) systems developed in our laboratory using a recombinant Der p 2 as target antigen. Sera from 240 children with mite allergy and 25 controls were measured.
Results The serum levels of specific IgE and, to lesser degree, lgG4 were higher in allergic children than non-allergic controls, while in the levels of the other IgG subclasses there was no difference between the two groups. There was no correlation between levels of specific IgF and IgG4 or in those between specific IgG4 and other IgG subclasses.
Conclusion Results indicate that the induction of Der p 2-specific lgG4 in allergic diseases is independent to IgE as well as other IgG subclasses.     

Reduction in humidity as a method of controlling mites and mite allergens: the use of mechanical ventilation in British domestic dwellings

Background Low humidity is an important limiting factor for mite population growth. Reducing humidity can therefore be used as a method to control mites within the home. Objective Ihis study investigated the effect of mechanical ventilation heat recovery (MVHR) units on house dust mites and mite allergen Der p I in typical homes in the North-West of England. Methods Mite counts and Der p 1 levels were measured at 3-monthly intervals over a period of 1 year in 18 houses (nine with MVHR units and nine architecturally matched control houses). Paired dust samples were collected using a vacuum cleaner with an air-flow rate 451/sec, adapted to collect the sample onto a preweighed filter paper. A 1 m² area of bedroom carpet, living room carpet and mattress was sampled for 2min. Indoor temperature and relative humidity (RH) levels were recorded for a period of 1 week before and after the winter period (November and February: 3 and 6 months data sets). The environmental questionnaire was completed at the beginning and at the end of the study. Results No difference in either Der p 1 concentrations or mite counts in any of the sampling sites at 3, 6, 9 and 12 months as compared with the baseline values was found, both within and between the groups (P > 0.1). The measured levels of RH performed in autumn and winter were found to be lower in the MVHR houses compared to the architectural controls. The indoor temperature during each period did not differ between the groups. Questionnaire data showed that the severity of condensation improved in the MVHR homes, whilst during the winter period, the severity of condensation had increased in the architectural control group. Conclusions The MVHR unit does not reduce indoor humidity to levels capable of retarding mite population growth and decreasing mile allergens in the type of houses predominantly found in the mild and humid climate of the North-West of England.     

Mouse/human chimeric IgGl and IgG4 antibodies directed to the house dust mite allergen Der p 2: use in quantification of allergen specific IgG

Background and Objective Chimeric mouse/human monoclonal IgGl and IgG4 antibodies were developed against the house dust mite allergen Der p 2. These chimeric IgG antibodies, hIgG1-Dp2 A and hIgG4-Dp2 A, have the same binding characteristics as the previously reported chimeric hIgE-Dp2 A and are composed of the heavy chain variable domains and light chains ot the original murine monoclonal antibody 2B12., whereas the heavy chain constant domains have been replaced by the human IgGl or IgG4 heavy chain. The expression level of hIgG1-Dp2 A and hIgG4-Dp2 A was 1 and 3.5 μg/mL, respectively. Methods and Results Since all IgG in these culture supernatants is allergen-specific. they are useful reference reagents and enable the calculation of the amount of allergen specific IgG l and IgG4 antibodies in absolute IgG amounts. The results obtained with two panels of sera from patients in immunotherapeutic treatment were evaluated and compared in Der p 2 IgE, IgGl and IgG4 RAST and with reversed lgG4 RAST using labelled purified Der p 2. Close agreement between the results for the two IgG4 assays was found. Conclusion With these chimeric reference reagents the quantities of isotype specific antiallergen antibodies can be calculated and compared.