A novel peanut allergy immunotherapy: Plant-based enveloped Ara h 2 Bioparticles activate dendritic cells and polarize T cell responses to Th1.

Introduction: As the only market-authorized allergen immunotherapy (AIT) for peanut allergy is accompanied by a high risk of side effects and mainly induces robust desensitization without sustained efficacy, novel treatment options are required. Peanut-specific plant-derived eBioparticles (eBPs) surface expressing Ara h 2 at high density have been shown to be very hypoallergenic. Here, we assessed the dendritic cell (DC)-activating and T cell polarization capacity of these peanut-specific eBPs. Methods: Route and kinetics of eBP uptake were studied by (imaging) flow cytometry using monocyte-derived DCs incubated with fluorescently-labelled Ara h 2 eBPs or natural Ara h 2 (nAra h 2) in the presence or absence of inhibitors that block pathways involved in macropinocytosis, phagocytosis, and/or receptor-mediated uptake. DC activation was monitored by flow cytometry (maturation marker expression) and ELISA (cytokine production). T cell polarization was assessed by co-culturing DCs exposed to Ara h 2 eBPs or nAra h 2 with naïve CD4+ T cells, followed by flow cytometry assessment of intracellular IFNγ+ (Th1) and IL-13+ (Th2), and CD25+CD127-Foxp3+ regulatory T cells (Tregs). The suppressive activity of Tregs was tested using a suppressor assay. Results: Ara h 2 eBPs were taken up by DCs through actin-dependent pathways. They activated DCs demonstrated by an induced expression of CD83 and CD86, and production of TNFα, IL-6, and IL-10. eBP-treated DCs polarized naïve CD4+ T cells towards Th1 cells, while reducing Th2 cell development. Furthermore, eBP-treated DCs induced reduced the frequency of Foxp3+ Tregs but did not significantly affect T cell IL-10 production or T cells with suppressive capacity. In contrast, DC activation and Th1 cell polarization were not observed for nAra h 2. Conclusion: Ara h 2 eBPs activate DCs that subsequently promote Th1 cell polarization and reduce Th2 cell polarization. These characteristics mark Ara h 2 eBPs as a promising novel candidate for peanut AIT.

Design, production and immunomodulatory potency of a novel allergen bioparticle.

Allergen immunotherapy (AIT) is the only disease-modifying treatment with evidence for sustained efficacy. However, it is poorly developed compared to symptomatic drugs. The main reasons come from treatment duration implying monthly injections during 3 to 5 years or daily sublingual use, and the risk of allergic side-effects. To become a more attractive alternative to lifelong symptomatic drug use, improvements to AIT are needed. Among the most promising new immunotherapy strategies is the use of bioparticles for the presentation of target antigen to the immune system as they can elicit strong T cell and B cell immune responses. Virus-like particles (VLPs) are a specific class of bioparticles in which the structural and immunogenic constituents are from viral origin. However, VLPs are ill-suited for use in AIT as their antigenicity is linked to structure. Recently, synthetic biology has been used to produce artificial modular bioparticles, in which supramolecular assemblies are made of elements from heterogeneous biological sources promoting the design and use of in vivo-assembling enveloped bioparticles for viral and non-viral antigens presentation. We have used a coiled-coil hybrid assembly for the design of an enveloped bioparticle (eBP) that present trimers of the Der p 2 allergen at its surface, This bioparticle was produced as recombinant and in vivo assembled eBPs in plant. This allergen biotherapeutic was used to demonstrate i) the capacity of plants to produce synthetic supramolecular allergen bioparticles, and ii) the immunomodulatory potential of naturally-assembled allergen bioparticles. Our results show that allergens exposed on eBPs induced a very strong IgG response consisting predominantly of IgG2a in favor of the TH1 response. Finally, our results demonstrate that rDer p 2 present on the surface of BPs show a very limited potential to stimulate the basophil degranulation of patient allergic to this allergen which is predictive of a high safety potential.

Dehydrin variants associated with superior freezing tolerance in alfalfa (Medicago sativa L.).

A cDNA (msaCIG) encoding a cold-inducible Y(2)K(4) dehydrin in alfalfa (Medicago sativa spp. sativa) was shown to share extensive homology with sequences from other species and subspecies of Medicago. Differences were mainly the result of the occurrence of large indels, amino acids substitutions/deletions and sequence duplications. Using a combination of a bulk segregant analysis and RFLP hybridization, we uncovered an msaCIG polymorphism that increases in frequency in response to recurrent selection for superior freezing tolerance. Progenies from crosses between genotypes with (D+) or without (D-) the polymorphic dehydrin significantly differed in their tolerance to subfreezing temperatures. Based on the msaCIG sequence, we looked for intragenic variations that could be associated to the polymorphism detected on Southern blots. Amplifications with primers targeting the 3' half side of msaCIG revealed fragment size variations between pools of genotypes with (+) or without (-) the polymorphism. Three major groups of amplicons of approximately 370 nt (G1), 330 nt (G2), and 290 nt (G3) were distinguished. The G2 group was more intensively amplified in pools of genotypes with the polymorphic dehydrin and was associated to a superior freezing tolerance phenotype. Sequences analysis revealed that size variation in the 3' half was attributable to the variable occurrence of large indels. Single amino acid substitutions and/or deletions caused major differences in the prediction of the secondary structure of the polypeptides. The identification of dehydrin variants associated to superior freezing tolerance paves the way to the development of functional markers and the fixation of favorable alleles in various genetic backgrounds.