Wan Daniel, Ludolf Fernanda, Alanine Daniel G W, Stretton Owen, Ali Ali Eman, Al-Barwary Nafal, Wang Xiaowei, Doenhoff Michael J, Mari Adriano, Fitzsimmons Colin M, Dunne David W, Nakamura Ryosuke, Oliveira Guilherme C, Alcocer Marcos J C, Falcone Franco H
Division of Molecular and Cellular Science, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom; School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
Genomics and Computational Biology Group, Centro de Pesquisas René Rachou, National Institute of Science and Technology in Tropical Diseases, Fundação Oswaldo Cruz - FIOCRUZ, Belo Horizonte, Minas Gerais, Brazil.
PLoS Negl Trop Dis. 2014 Sep 25;8(9):e3124. doi: 10.1371/journal.pntd.0003124. eCollection 2014 Sep.
Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.
METHODOLOGY/PRINCIPAL FINDINGS: A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.
CONCLUSION/SIGNIFICANCE: This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.
寄生虫特异性IgE被认为与抵御曼氏血吸虫感染或再感染相关。在曼氏血吸虫感染中，IgE反应的分子靶点仅有少数得到了表征。通过对这类曼氏血吸虫变应原进行全基因组表征，能够更深入地了解抗寄生虫免疫的基本机制。这将对我们理解过敏以及开发安全有效的抗蠕虫寄生虫疫苗产生影响。
方法/主要发现：本文描述了一个完整的适用于中高通量的工作流程，包括重要的质量控制环节，该流程能够利用小麦胚裂解物快速翻译曼氏血吸虫蛋白，并随后使用人源化大鼠嗜碱性白血病（RBL）报告细胞系评估潜在变应原性。无细胞翻译在90分钟内完成，产生足够量的寄生虫蛋白用于快速筛选变应原性，无需任何纯化步骤。通过蛋白质免疫印迹法证明抗原完整性。在用感染者血清过夜孵育后，用完整的小麦胚翻译混合物刺激RS-ATL8报告细胞系，并测量荧光素酶活性，以报告可疑变应原引起的细胞活化情况。使用在小麦胚裂解物和/或大肠杆菌中表达的特征明确的植物和寄生虫变应原，如Par j 2、SmTAL-1和IgE结合因子IPSE/alpha-1，证明了该系统用于表征新型曼氏血吸虫变应原的适用性。SmTAL-1能够激活嗜碱性粒细胞报告细胞系，而SmTAL2（用作阴性对照）则不能。
结论/意义：该方法为评估抗蠕虫疫苗候选物的潜在变应原性提供了一种可行的途径，适用于使用感染者血清进行的中高通量研究。它也适用于研究蠕虫蛋白变应原性的基础。
