(549f) In Vitro Activation of Murine Dendritic Cells by Carbohydrate-Modified Polyanhydride Adjuvants

Design of single dose vaccines is a key strategy in providing immunological defense against infectious agents. Current strategies for the development of effective vaccines are based on the use of adjuvants and recombinant protein antigens with poor immunogenicity. Polyanhydride microparticles are promising platforms for use as vaccine delivery systems. These vehicles have been shown to: a) stabilize protein antigens; b) provide sustained and slow release of antigens; and c) enhance the adjuvant effect by modulating the immune response. Modification of these delivery vehicles with specific pure synthetic carbohydrates, to which essentially all humans will have pre-existing innate or adaptive immune response, has the potential to enhance their adjuvanticity. Dendritic cells (DCs) are antigen presenting cells that play a major role in connecting the innate and adaptive immune systems, which is a necessary step to induce protective immunity against infectious agents. The main goal of this work is to study the adjuvant effect provided by carbohydrate-modified polyanhydride microparticles and specifically understand how murine DCs uptake these particles. A non-aqueous cryogenic atomization method was used to fabricate polyanhydride microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH), sebacic acid (SA), and 1,8-bis(p-carboxyphenoxy)-3,6-dioaoctane (CPTEG) in various ratios. Di-mannose and lactose were attached to the surface of the polymer microparticles (which contain hydrophobic patterns) forming a surface that can be recognized by specific receptors on DCs (e.g., mannose receptor) and an unreactive carbohydrate surface, respectively. Scanning Electron Microscopy (SEM) and X-Ray Photoelectron Spectroscopy (XPS) were used to characterize the modified particles. DCs were cultured from the bone marrow of C57/BL6 mice and incubated with the carbohydrate-modified microparticles. After incubation, DCs were collected and assessed by flow cytometry for the surface expression of CD11c, MHC II, CD86, CD40, and CD209. In addition, culture supernatants were collected and analyzed for the presence of cytokines (IL-6, IL-10, IL-12p40, and TNF-alfa). DCs incubated with lactose-modified particles displayed enhanced expression of all cell surface markers and production of TNF-alfa compared to DCs stimulated with unmodified particles, while the the presence of di-mannose appeared to have an inhibititory effect on both cells surface marker expression and cytokine production. This suggests that surface modification by specific carbohydrates can allow activation of immune pathways. These studies provided new insights on how adjuvants initiate immune responses, and the knowledge gained can be used to effectively and rationally design new vaccines against known and emerging pathogens.