Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Abstract

BackgroundParasite-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 FindingsA 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/SignificanceThis 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.

Of Monkeys and Men: Immunomic Profiling of Sera from Humans and Non-Human Primates Resistant to Schistosomiasis Reveals Novel Potential Vaccine Candidates

“…IgE poses somewhat of a conundrum for helminth vaccinologists due to its clear association with naturally acquired protection ( 22 , 63 ), but the accompanying risk of vaccinating people with a recombinant protein that is the target of pre-existing IgE responses and poses the risk of inducing atopy ( 64 ), or potentially anaphylaxis. Instead of excluding potentially protective IgG1-inducing antigens that are the targets of parasite-derived IgE in exposed individuals from further vaccine development, we propose that the molecules be assessed for allergenicity through the use of basophil-activation studies, given that the induction of IgE and clinical manifestation of allergy are not mutually inclusive events ( 94 ). Another strategy aimed at minimizing potential allergenicity of helminth proteins involves their fusion to Fcγ, thereby directing the chimeric protein to the negative signaling receptor FcγRIIb expressed on pro-allergic cells ( 95 ).…”

RBL cells as models for in vitro studies of mast cells and basophils

“…This sensitive RS‐ATL8 assay has since been used for the detection of food or other allergens and is amenable to high throughput format . It has also been suggested for assessment of allergenicity of anti‐schistosome vaccine candidates and for other purposes, as described recently by us …”

IPSE, a parasite-derived host immunomodulatory protein, is a promising therapeutic for hemorrhagic cystitis

“…We previously demonstrated that M-IPSE activates basophils in vitro through NF-AT (23). This pathway is implicated in basophil and mast cell expression of IL-4, which we have observed in vivo in mice administered H-IPSE H06 (17).…”

“…Basophil activation with recombinant M-IPSE, H-IPSE H03 and H-IPSE H06 Basophil activation was quantified as previously described (23). RS-ATL8 cells were cultured in 10 mL MEM (GIBCO, USA), supplemented with 5% vv v/v heat-inactivated FCS (GIBCO, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma, UK) and 2 mM L-glutamine (Sigma, UK).…”