Project Summary/Abstract Although numerous biological and pharmaceutical agents are known to promote advantageous cellular responses, the targeted, localized delivery of immunomodulating factors to specific cellular and subcellular targets remains a challenge for the development of safe, efficacious modulation of the immune system. An attractive strategy involves the in vivo administration of antigen encapsulated in surface-modified polymeric microparticles (MPs) as a vaccine targeted to dendritic cells (DCs). Micron-sized particles are phagocytosable, effectively targeting phagocytes over non-phagocytes by virtue of their particulate form, and can be further targeted to DCs, the most efficient antigen presenting cell type of the body and key regulator of the immune system. A MP-based vaccine may be easily administered and can deliver both prime &boost doses using biodegradable time-release materials. Therapeutic vaccination approaches for type-1 diabetes utilizing DC-targeting MPs hold promise to correct antigen-specific autoimmune responses. The objective of this proposal is to engineer a subcutaneously injectable two-MP vaccine system consisting of i.) DC-targeting phagocytosable MPs delivering insulin peptide antigen and vitamin D3 to intracellular targets and ii.) non-phagocytosable MPs to deliver sustained, localized to the injection site, DC recruitment and immuno-suppressive biological factors (GM-CSF and TGF-&#61538;1) to effect a pro-tolerogenic DC phenotype promoting induction of regulatory T-cells, antigenspecific suppression of auto-reactive T-cells, and prevent diabetes in NOD mice. The hypothesis is that a multi-component two-microparticle system having both intracellular and cell-surface receptor targets will more effectively provide robust, durable antigen-specific immune suppression than each single-component formulation separately or in soluble bolus form. Specific Aim 1 is to formulate the multi-component two-MP system and test it in vitro, characterizing DC phenotype (activated, immature or tolerogenic) and T-cell response (stimulation, Th1, Th2, Treg, or Th17). Specific Aim 2 is to evaluate the ability of the two-MP formulation in vivo, aiming to prevent diabetes in NOD mice. The two-MP system is novel and innovative as it represents a simply administrable vaccine formulation that simultaneously provides targeted delivery of antigen and hydrophobic intracellularly-acting agent to DC intracellular compartments, while extracellularly delivering DC recruitment and immunomodulatory factors to their associated cell-surface receptors.