ASSOCIATE PROFESSOR University of Florida, United States
Introduction:: Roughly 15 million Americans suffer from food allergies and typically manage their symptoms through strict food avoidance and/or the adminstration of antihistamine upon exposure. Presently, oral immunotherapy (OIT) is the most efficacious option to achieve sustained unresponsiveness (SU) in allergic patients but is limited by the risk of anaphylaxis. Several clinical studies indicate that SU after OIT is correlated with increased T regulatory (Treg) cell populations, which suggests the supplementation of OIT with tolerogenic factors, such as gut-derived commensal molecule Polysaccharide A (PSA), will increase its efficacy. We hypothesize that the encapsulation of allergen within PSA nanoparticles (NPs) will show significant improvements in efficacy and safety over traditional allergen-only oral immunotherapy due to PSA NPs ability to (i) induce Treg differentiation and deliver allergen simultaneously, and (ii) to shield the allergen from IgE receptor-mediated mast cell activation until internalization by intestinal dendritic cells (Figure 1). We show that PSA NPs can be fabricated using water/oil emulsification with chemical crosslinking and maintain immunoregulatory capability while delivering protein antigen (Figure 2). PSA NPs have the potential to become a “plug-in-play” system to induce specific tolerance to any encapsulated antigen.
Materials and Methods:: PSA NPs were fabricated using water/oil emulsification with chemical crosslinking. Morphology and size were analyzed with scanning electron microscopy (SEM) and dynamic light scattering (DLS). HEK-Blue mTLR2 reporter cells (Invivogen) were treated with 25ug/ml PSA, PSA NPs, or OVA-loaded PSA-NPs (PSA-OVA NPs) for 16 hours in the absence or presence of blocking anti-mTLR2 antibody. Flt3L BMDCs were incubated with 50ug/ml PSA, PSA NPS, or PSA-OVA NPs for 2 days before flow cytometry analysis.CD4+ T cells and Flt3L BMDCs (5:1) from OTII mice were incubated with 50ug/ml PSA, PSA NPs, or PSA-OVA NPs for 4 days before performing flow cytometry analysis and IL-10 ELISA.
Results, Conclusions, and Discussions:: Our results indicate that ova-loaded PSA NPs can be fabricated using water/oil emulsification with glutaraldehyde crosslinking. PSA-OVA NPs successfully delivers OVA to dendritic cells and induces downstream tolerogenic phenotypes in DCs and OVA-specific T cells and IL-10 production. To our knowledge, our data demonstrates the first successful method of generating protein-loaded PSA nanoparticles. In future experiments, PSA NPs will be loaded with tropomyosin, the model allergen for shrimp allergy, and tested against sera and cells derived from shellfish-allergic paitents to determine PSA NP’s ability to (i) block binding from allergen-specific IgE, and (ii) prevent degranulation and histamine release from sensitized mast cells. If successful, an induced-shrimp allergy mouse model will be used to evaluate PSA NP’s ability to augment OIT.
