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- Title
Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins.
- Authors
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.
- Abstract
Background: 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. Author Summary: Infection with parasitic helminths is characterised by a marked elevation of total and parasite-specific Immunoglobulin E (IgE). It is widely believed that this IgE response has evolved to protect hosts against large metazoan parasites. Such a protective function has been well characterised in particular against members of the genus Schistosoma. However, with a few notable exceptions, the molecular targets of the IgE response and the downstream immunological mechanisms leading to host protection are not well understood. The molecular targets of a specific IgE response are by definition called allergens. While almost 3,000 different allergens, contained in e.g. plant pollen or seeds, moulds or animal materials, have been characterised at the molecular level, and are listed and described in databases such as the Allergome database (www.allergome.org), only a few dozen allergens have been characterised in parasitic helminths. A more detailed understanding of the molecular targets of the anti-helminth IgE response can not only be expected to further our basic understanding of protective immune responses and allergy in general–such knowledge can also be expected to have important repercussions on the production of safe and effective anti-helminthic vaccines. This research describes a novel approach suitable for genome-wide functional identification of allergens in S. mansoni and other parasites, paving the way for the identification of the Schistosoma allergome.
- Subjects
SCHISTOSOMA mansoni; CELL lines; WHEAT germ; ESCHERICHIA coli; IMMUNOGLOBULIN E; PARASITOLOGY; MALE sterility in plants
- Publication
PLoS Neglected Tropical Diseases, 2014, Vol 8, Issue 9, p1
- ISSN
1935-2727
- Publication type
Article
- DOI
10.1371/journal.pntd.0003124